Novel Compositions and Methods for Treating Cancer

ABSTRACT

Provided herein are methods and compositions inter alia for treating diseases, including hyperproliferative diseases, migraine headaches, and depression.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/662,135, filed Oct. 26, 2012, which in turn claims the benefit ofU.S. Provisional Patent Application No. 61/553,051, filed Oct. 28, 2011,which is incorporated herein by reference in its entirety and for allpurposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 48932-510C01US_ST25.TXT, created onApr. 28, 2015, 1,791 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Breast Cancer is the most common malignancy in women in the Westernworld and every year about 200,000 women are diagnosed with breastcancer in the US and more than 40,000 die from this disease(www.cdc.gov). Despite the fact that combination chemotherapy regimenselicit a 50-70% objective response rate in patients with metastaticbreast carcinoma less than 20% of patients achieve durable completeremission (Esteva F J, et al. J Clin Oncol 2002: 20:1800-8). The majorreason for patient death is due to metastasis and resistance to currenttherapies including chemotherapy, hormonal therapy and radiation(Polychemotherapy for early breast cancer: an overview of the randomisedtrials. Early Breast Cancer Trialists' Collaborative Group. Lancet 1998:352:930-42.). Thus, the development of novel targeted therapeuticstrategies is urgently needed to enhance the efficacy of currenttherapies and prolong patient survival.

Calmodulin-dependent protein Kinase-III (CaMK-III) a.k.a. Eukaryoticelongation factor 2 kinase (eEF-2K) is an unusual calcium/calmodulin(Ca/CaM)-dependent Ser/Thr-kinase that is activated by mitotic agentsinvolved in cell proliferation and viability (Parmer T G, et al. Br JCancer 1999: 79:59-64). CaMK-III was originally identified as a Ca²⁺/CaMdependent protein kinase (Nairn A C, et al. Proc Natl Acad Sci USA 1985:82:7939-43). It is highly regulated by second messengers (e.g. Ca²⁺,PIP₃), as well as by a number of different protein kinases. Uponmitogenic stimulation there is a rapid activation of CaMK-III, whichleads to the subsequent phosphorylation of elongation factor 2 (eEF2),which regulates elongation of protein synthesis (Parmer T G, et al. CellGrowth Differ 1997: 8:327-34).

CaMK-III activity has been found to be significantly increased in breastcancer specimens but absent in normal tissue adjacent to breast cancer(Chafouleas J G, et al. Proc Natl Acad Sci USA 1981: 78:996-1000).Increased activity of this kinase has been reported in proliferatingcells (e.g. malignant glioma cells (Bagaglio D M, Hait W N. Cell GrowthDiffer 1994: 5:1403-8), and HL60 leukemia cells (Nilsson A, Nygard O.Biochim Biophys Acta 1995: 1268:263-8)), but is reported to be absent innonproliferating cells (Parmer T G, et al. Cell Growth Differ 1997:8:327-34). In breast cancer cells, the activity of CaMK-III isstimulated by mitogens and growth factors (Parmer T G, et al. Activityand regulation by growth factors of calmodulin-dependent protein kinaseIII (elongation factor 2-kinase) in human breast cancer. Br J Cancer1999: 79:59-64). Progression through the G1-phase of the cell cycle andentry into the S phase (the G1/S transition) requires the activity ofCaMK-III, which is mediated by a rise in intracellular calcium (Ca²⁺),and/or the up-regulation of c-AMP (Proud C G. Biochem J2007:403:217-34). Hypoxia, nutrient deprivation and metabolic stressstimulate CaMK-III through activation of AMPK, leading to thephosphorylation of eEF2 and the inhibition of protein synthesis (BrowneG J, et al. J Biol Chem 2004: 279:12220-31).

Serotonin (5-hydroxytryptamine, 5-HT), a monoamine neurotransmitter, isa critical local regulator of epithelial homeostasis in the breast andother organs. Serotonin exerts its actions through a repertoire of 15 ormore receptor proteins, belonging to seven discreet families. Six of thefamilies of 5-HT receptors are G-protein-coupled, including G_(i):5-HT₁, Gs: 5-HT_(4,6,7), and G_(q/11): 5-HT_(2,5). 5-HT₃ is uniquely aligand-gated cation channel, related to the nicotinic acetylcholinereceptor. Previous studies suggest that serotonin, plays a mitogenicrole in cancer cells including bladder, pancreatic, prostatehepatocellular cancers, small cell lung carcinoma cells [Parmer T G, etal. Br J Cancer 1999: 79:59-64, Nairn A C, et al. Proc Natl Acad Sci USA1985: 82:7939-43, Parmer T G, et al. Cell Growth Differ 1997: 8:327-34,Chafouleas J G, et al. Proc Natl Acad Sci USA 1981: 78:996-1000,Bagaglio D M, Hait W N. Cell Growth Differ 1994: 5:1403-8, Nilsson A,Nygard O. Biochim Biophys Acta 1995: 1268:263-8] and breast cancer(Sonier et al., 2006). Among the 5-HT receptors, the 5-HTR2B has beendescribed to mediate proliferation [Proud C G. Biochem J2007:403:217-34, Browne G J, et al. J Biol Chem 2004: 279:12220-31, Franken NA, et al. Nat Protoc 2006: 1:2315-9] and recently, 5-HTR2A signaling hasbeen shown to promote mitogenic signal in MCF7 breast cancer cells [Lu KP, Means A R. Endocr Rev 1993: 14:40-58]. It has been reported thatcomplex alterations in the intrinsic mammary gland serotonin system ofhuman breast cancers exist [Liao D J, Dickson R B. Endocr Relat Cancer2000: 7:143-64]. Pai et al. demonstrated that in the normal mammarygland, 5-HT acts as a physiological regulator of lactation andinvolution, in part by favoring growth arrest and cell death. Thistightly regulated 5-HT system is dysregulated in multiple ways in humanbreast cancers. Specifically, tyrosine hydroxylase, TPH1, (an enzymefound in peripheral tissues leads to production of serotonin andexpressed in non neuronal tissues) expression increases during malignantprogression. 5-HT receptor expression is dysregulated in human breastcancer cells, with increased expression of some isoforms and suppressionof others. The receptor expression change is accompanied by altereddownstream signaling of 5-HT receptors in human breast cancer cells,resulting in resistance to 5-HT-induced apoptosis, and stimulatedproliferation. Pai et al found that HT1D, 1F, 2C and 3A are expressed insome breast cancer cells compared to MCF10A normal breast epithelium[Liao D J, Dickson R B. Endocr Relat Cancer 2000: 7:143-64].

Apoptosis (programmed cell death type I) and autophagic cell death(programmed cell death type II) are crucial physiological mechanismsthat control the development, homeostasis, and elimination of unwantedand malignant cells [Musgrove E A. Growth Factors 2006: 24:13-9]. It isnow evident that elimination of cancer cells following chemotherapytreatment occurs, in part, via the induction of autophagic cell death[Abukhdeir A M, Park B H. Expert Rev Mol Med 2008;:10:e19, Ozpolat B,Mol Cancer Res 2007: 5:95-108, Shaw L M. Methods Mol Biol 2005:294:97-105], Autophagic cell death or type II programmed cell death is aform of non-apoptotic cell death that can be induced by differentconditions including serum starvation, gamma-radiation, toxic stimuli,and chemotherapy [Musgrove E A. Growth Factors 2006: 24:13-9].

Autophagy is characterized by an increase in the number ofautophagosomes, vesicles that surround such cellular organelles as Golgicomplexes, polyribosomes, and the endoplasmic reticulum [Finn R S. AnnOncol 2008: 19:1379-86]. Subsequently, autophagosomes merge withlysosomes and digest the organelles, leading to cell death. In contrastto apoptosis, autophagic cell death does not involve classic DNAladdering. A growing body of evidence suggests that alterations in thepathways regulating autophagic cell death may result in cancerdevelopment. Provided herein are methods and compositions addressingthese and other needs in the art.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, a compound is provided having the formula:

R^(1A) is independently hydrogen, halogen, —CX^(1A) ₃, —C(O)R^(7A),—C(O)—OR^(7A), —C(O)NR^(7A)R^(8A), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.R^(1B) is independently hydrogen, halogen, —CX^(1B) ₃, —C(O)R^(7B),—C(O)—OR^(7B), —C(O)NR^(7B)R^(8B), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.R^(2A) is independently hydrogen, halogen, —CX^(2A) ₃, —C(O)R^(9A),—C(O)—OR^(9A), —C(O)NR^(9A)R^(10A), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.R^(2B) is independently hydrogen, halogen, —CX^(2B) ₃, —C(O)R^(9B),—C(O)—OR^(9B), —C(O)NR^(9B)R^(10B), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.R^(1A) and R^(2A) are optionally joined to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl. R^(1B) and R^(2B) are optionally joined to form asubstituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl. R³ is independently hydrogen, halogen, —CX³ ₃,—CN, —SO₂Cl, —SO_(n)R¹⁴, —SO_(k)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹²,—NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹², —N(O)_(m), —NR¹¹R¹², —C(O)—OR¹³,—O—C(O)—R¹³, —C(O)NR¹¹R¹², —NR¹¹C(O)R¹³, —OR¹⁴, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R^(7A), R^(7B), R^(8A), R^(8B), R^(9A),R^(9B), R^(10A), R^(10B), R¹¹, R¹², R¹³, and R¹⁴ are independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Thesymbols A¹ and A² are independently ═N— or ═CR³—. The symbol L isindependently a bond, —O—, —NH—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, substitutedor unsubstituted heteroarylene,

or —O—(CH₂)_(p)—O—. The symbols k and m are independently 1 or 2. Thesymbol n is independently an integer from 0 to 4. The symbols p, v, andw are independently an integer from 1 to 20. The symbols z1 and z2 areindependently an integer from 0 to 3. The symbols X^(1A), X^(1B),X^(2A), X^(2B), and X³ are independently —Cl, —Br, —I, or —F.

In a second aspect, a pharmaceutical composition is provided thatincludes a pharmaceutically acceptable excipient and a compound asdescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe),(If), (IIIf), (Ig), (IIIg), (Ih) or (IIIh), including embodiments, orany compound described in the Examples section herein).

In a third aspect, a method of treating a disease in a patient in needof such treatment is provided. The method includes administering atherapeutically effective amount of a compound as described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig),(IIIg), (Ih) or (IIIh), including embodiments, or any compound describedin the Examples section herein).

In another aspect, a pharmaceutical composition is provided thatincludes a pharmaceutically acceptable excipient and an anti-eEF-2Kinhibitory nucleic acid, anti-5-HTR_(1B) inhibitory nucleic acid,anti-5-HTR_(1D) inhibitory nucleic acid, or an anti-5-HTR inhibitorynucleic acid.

In another aspect, a method of treating a disease in a patient in needof such treatment is provided. The method includes administering atherapeutically effective amount of an anti-eEF-2K inhibitory nucleicacid, anti-5-HTR_(1B) inhibitory nucleic acid, anti-5-HTR_(1D)inhibitory nucleic acid, or an anti-5-HTR inhibitory nucleic acid.

In another aspect, a method of treating a disease in a patient in needof such treatment is provided. The method includes administering atherapeutically effective amount of a pharmaceutical composition asdescribed herein (e.g. including a compound of Formula (I), (II), (III),(IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb),(Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd),(Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih) or (IIIh),including embodiments, or any compound described in the Examples sectionherein).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. An example of a targeted long circulating liposome. RGD-modified(αVβ3 integrin receptor targeted) long circulating liposomes fortumor-specific compound (e.g. triptan) delivery.

FIG. 2. Synthesis of a library of triptan analogs.

FIG. 3. New role for triptan derivatives targeting the 5-HT_(1B)receptor as a therapeutic modality for breast cancer. (A). In vivotargeting of the 5-HT_(1B/1D) receptor by neutral lipid-based(DOPC)-liposomal 5-NT in nude mice bearing MDA-MB-231 breast cancerxenografts significantly inhibits tumor growth. Treatments, twice perweek (4 weeks) were safe and effective. Methods: Phospholipiddioleoylphosphatidylcholine (DOPC) and triptan 5-NT were mixed intertiary butanol at a lipid:5-NT ratio of 10:1 (w/w). Tween 20 was addedat a ratio of 1:20 ratio (w/w) and the mixture lyophilized overnight.The powder was reconstituted in saline and sonicated for one minute. Theliposome formulation was injected (100 μl) into the tail vein. (B). 5-NTinduces apoptotic cell death of ER(+) MCF-7 cells. (C). 5-NT inhibitscolony formation of ER(+) MCF-7 cells. (D). 5-nonylytryptamine (5-NT)(Glennon et al., 1996) inhibits proliferation of triple negativeMDA-MB-231, as well as ER(+) MCF-7 and MCF7/Dox resistant cells. (E).The 5-HT_(1B) receptor is over-expressed in triple negative highlyaggressive, and metastatic breast cancer cells as well as drug resistant(i.e. tamoxifen and doxorubicin) breast cancer cells. Methods:Blots werevisualized with a FluorChem 8900 imager and quantified by a densitometerusing the Alpha Imager application program.

FIG. 4. eEF-2K regulates the activation of IGF-1R, Akt, and mTOR throughSrc in MDA-MB-231 cells. Western blot analysis shows that eEF-2K siRNAinhibits eEF-2K expression. This leads to the concomitantdown-regulation of Src activity (as determined by detection of phosphoTyr-416). The down-regulation of eEF-2K or Src leads to thedown-regulation of IGF-1R and Akt activity. Methods:Cells weretransfected by eEF-2K or Src siRNA (75 nM) for 48 h and assessed byWestern blotting as in FIG. 5.

FIG. 5. 5-NT down-regulates critical pro-tumorigenic processes in ER(+)MCF-7 triple negative breast cancer cells. Western blot analysis showsthat 5-NT (5 μM) down-regulates the expression of eEF-2K, Cyclin D,NF-KB, and VEGF as well as the phosphorylation of IGF1R, Src, FAK, mTOR,p70S6K, and ERK2. 5-NT also induces PARP cleavage and LC3-II. Methods:MCF7 cells were cultured at 37° C. in DMEM supplemented with 5% FBS in ahumid incubator with 5% CO₂. Following incubation with or without 5-NTcells were lysed according to standard protocols for western blotting.Blots were quantified as in FIG. 3.

FIG. 6. New role for triptans as a therapeutic modality for breastcancer. (A). In vivo targeting of the 5-HT_(1B/1D) receptor by neutrallipid-based (DOPC)-liposomal 5-nonylytryptamine (5-NT) (Glennon et al.,1996b) in nude mice bearing MDA-MB-231 breast cancer xenograftssignificantly inhibits tumor growth. Treatments, twice per week (4weeks) were safe and effective. Methods: Phospholipiddioleoylphosphatidylcholine (DOPC) and triptan 5-NT were mixed intertiary butanol at a lipid:5-NT ratio of 10:1 (w/w). Tween 20 was addedat a ratio of 1:20 ratio (w/w) and the mixture lyophilized overnight.The powder was reconstituted in saline and sonicated for one minute. Theliposome formulation was injected (100 μl) into the tail vein. (B). 5-NTinduces apoptotic cell death of ER(+) MCF-7 cells. (C). 5-NT inhibitscolony formation of ER(+) MCF-7 cells. (D). 5-NT inhibits proliferationof triple negative MDA-MB-231, as well as ER(+) MCF-7 and MCF7/Doxresistant cells. (E). 5-NT shows much higher selectivity towards thetumorigenic cell line MCF-7, when compared to MCF-10A, a non-tumorigeniccell line, and appears to be non-toxic to mice.

FIG. 7. siRNA targeting of eEF-2K significantly inhibits tumor growth.(A). Therapeutic targeting of eEF-2K by systemically administeredliposomal siRNA inhibits orthotopic tumor growth in a breast cancermodel. Methods: About 2 weeks after injection of MDA-MB-231 cells(1×10⁶) into mammary fat pat (orthotopic), therapeutic targeting of theeEF-2K gene was achieved by systemically (i.v.) administeredDOPC-nanoliposomal siRNA (L-eEF-2K siRNA) (150 μg/kg or about 4μg/mouse, twice a week for 4 weeks). Nonsilencing DOPC-liposomal siRNA(L-Control siRNA) was used as control. Two different eEF-2K-targetingsiRNAs were used; eEF-2K siRNA(#1) and siRNA(#2). At the end of week 4,the experiment was terminated and tumor weight was measured. (B). eEF-2Ksilencing inhibits proliferation triple negative MDA-MB-231 cells. (C).eEF-2K silencing inhibits colony formation of triple negative MDA-MB-231cells. (D). eEF-2K silencing inhibits invasion/migration of triplenegative MDA-MB-231. (E). eEF-2K is overexpressed across a variety ofbreast cancer cell lines. Methods: Blots were visualized with aFluorChem 8900 imager and quantified by a densitometer using the AlphaImager application program.

FIG. 8. eEF-2K regulates the activation of IGF-1R, Akt, and mTOR throughSrc in MDA-MB-231 cells. Western blot analysis shows that eEF-2K siRNAinhibits eEF-2K expression. This leads to the concomitantdown-regulation of Src activity (as determined by detection of phosphoTyr-416). The down-regulation of eEF-2K or Src leads to thedown-regulation of IGF-1R, Akt and mTOR activity. Additionally FAK,NF-kB, VEGF, cyclin D1 and c-myc are downregulated, while p27 levelsincrease. Methods: Cells were transfected with eEF-2K or Src siRNA (75nM) for 48 h and assessed by Western blotting. Blots were quantified asin FIG. 7.

FIG. 9. 5-NT down-regulates critical pro-tumorigenic processes in ER(+)MCF-7 breast cancer cells. Western blot analysis shows that 5-NT (5 μM)down-regulates the expression of eEF-2K, Cyclin D1, NF-kB, and VEGF aswell as the phosphorylation of IGF-1R, Src, FAK, mTOR, p70S6K, and ERK2.5-NT also induces PARP cleavage and LC3-II formation. Methods: MCF7cells were cultured at 37° C. in DMEM supplemented with 5% FBS in ahumid incubator with 5% CO₂. Following incubation with or without 5-NT,cells were lysed according to standard protocols for western blotting.Blots were quantified as in FIG. 7.

FIG. 10. Possible mechanism for how triptan derivatives, such as 5-NTdisrupt tumorigenic signaling of the 5-HT_(1B) receptor. Binding of 5-NTto the 5-HT_(1B) receptor leads to the down-regulation of eEF-2Kexpression as well as the inactivation of c-Src and Fak, IGF-1R,Akt/mTor and Erk. These events may contribute to the observedpharmacological effects of 5-NT on breast cancer cells expressing adysregulated serotonin system. The mechanism in this figure is based onthe reported roles of the depicted genes in the literature, andrepresents a starting point from which to fully evaluate the mechanismof action of triptan derivatives.

FIG. 11. 5-NT and KD06 exhibit anti-tumor effects by signaling throughthe 5-HT_(1B/1D) receptor. (A). KD06, a triptan derivative that we havesynthesized, is more potent than 5-NT. (B). The 5-HT_(1B) receptor isover-expressed in triple negative highly aggressive, and metastaticbreast cancer cells as well as drug resistant (i.e. tamoxifen anddoxorubicin) breast cancer cells. (C). Knockdown of either the 5-HT_(1B)or 5-HT_(1D) receptor blunts the effect of 5-NT and KD06. (D). 5-NT andKD06 also decrease cAMP levels, which is known to be associated with5-HT_(1B/1D) receptor signaling.

FIG. 12. Characterization of compounds. Compound are numbered with KDnumbers Examples (e.g. Compound 1 in FIG. 12 corresponds to compoundKD01 in the Examples section, Compound 37 in FIG. 12 corresponds tocompound KD37 in the Examples section).

FIG. 13. (A). CaMK-III protein is overexpressed in a panel of breastcancer cells compared to non-tumorigenic normal breast epithelium(MCF10A). (B). A specific siRNA inhibits protein expression of CaMK-IIIin MDA-MB-231. Cells were transfected by CaMK-III siRNA (5-75 nM) for 48h and cell lysates were subjected to Western blot analysis. (C).Knockdown of CaMK-III inhibits proliferation of MDA-MB-231 cellsdetected by MTS assay (*p<0.05). (D). Knockdown of CaMK-III by siRNA (50nM) induces PARP cleavage (48 h). (E). CaMK-III overexpression increasescell proliferation of MDA-MB-231 cells. Cells were transientlytransfected with CaMK-III or a vector control and cell viability wasmeasured using an MTS assay at 96 h.

FIG. 14. (A) Knockdown of CaMK-III by siRNA (50 nM) significantlyinhibited number of colonies formed by MDA-MB-231 (B) and in MCF7cells(*p<0.05). (C). Cells were transtected with siRNA every 4 days. (D)Knockdown of CaMK-IIIsiRNA c-myc, cyclin D1 and increased the proteinexpression of the CDK-Inhibitor p27^(Kip1) (72 h). (E) The knockdown ofCaMK-III also reduced the expression of a transcriptionaly active formof NF-κB (p-p65-ser-356). (F) Depletion of CaMK-III by siRNA inhibitsinvasion of MDA-MB-231 cells in matrigel (G) Overexpression of CaMK-IIIincreases invasion of MDA-MB-231 cells in matrigel (72 h).

FIG. 15. Knockdown of CaMK-III by siRNA inhibits the activity of c-Src,Fak, PI3K/Akt and IGF-1R as indicated by reduced (A) p-Src (Thr-416),(B) p-Fak (Thr-397) (D) p-IGF1R (Tyr1131) in MDA-MB231 cells (72 h).Depletion of of CaMK-III also inhibits p-mTOR, p-Paxillin and VEGF.Knockdown Src by siRNA leads to reduction of Akt and IGF1R activity,suggesting that CaMK-III particiates regulation of AKT. Overexpressionof CaMK-III increases (C) p-Src and (E) p-IGFR in MDA-MB231 cells. Cellswere transiently transfected with GST-CaMK-III plasmid and 72 h latercells were collected for Western blot analysis. (F) Knockdown ofCaMK-III by siRNA inhibits the activity of p-Akt (Ser473).

FIG. 16. (A). Therapeutic targeting of the CaMK-III gene was achieved bysystemically (i.v.) administered DOPC-liposomal siRNA (L-CaMK-IIIsiRNA#1 and siRNA#2 [42]) or non-silencing DOPC-liposomal siRNA(L-Control siRNA) in nude mice bearing MDA-MB-231 tumors. At the end ofweek 4, tumor weight was measured. (B). Mice bearing MDA-MB231 tumorswere given L-CaMK-III siRNA or L-control siRNA (i.v, twice a week, 150μg/kg) and doxorubicin once a week (i.p, 2.5 mg/kg) for 4 weeks andtumor weight were measured. (C) Western blot analysis were performed intumor samples that were resected 48 h after the last siRNA injectionshowed down modulation target CaMK-III protein and induction apoptosisas indicated by caspase-9 cleavege and (D) TUNEL assay. (E)Quantification of TUNEL % positive cells in tumors.

FIG. 17. (A). Knockdown of CaMK-III induces Caspase-9 cleavege andincreases Doxorubicin-induced apoptosis in MCF7/DoxR cells. About 48hours after siRNA transfection cells were treated with doxorubicin andlysed for Western blot analysis. (B). Depletion of CaMK-III enhancesorubicin induced Caspase-9 cleavage in MDA-MB231 cells. (C). Proposedmechanism of CaMK-III-mediated pathways and biological endpoints inbreast cancer. CaMK-III is involved in cell proliferation, invasion andsurvival in breast tumors Inhibition of CaMK-III expression inducesapoptosis and activity of c-Src/Fak/paxillin as well as IGF1R andPI3K/Akt. CaMK-III expression is involved in regulation of cellproliferation, survival and angiogenesis by participating to theexpression c-myc, NF-κB, cyclinD1, Bcl-2 and VEGF and HIF1α expressionthat are associated with tumorigenesis. CaMK-III expression suppressesPDCD4, a tumor suppressor protein known to inhibit cellmigration/invasion and to induce p27^(Kip1) expression.

FIG. 18. Western blot analysis demonstrated that inhibition of CaMK-IIIin MDA-MB231 cells further reduced cyclin D1 was and increased thecycline-dependent kinase (CDK)-inhibitor p27^(Kip1) and PDCD4 tumorsuppressor protein, a regulator of p27^(Kip1), response to doxorubicin.

FIG. 19. (A). Knockdown of CaMK-III inhibits Src and FAK activity andincreases Doxorubicin-induced apoptosis in MCF7/DoxR cells. CaMK-IIIdepletion also inhibited p-EF2 (Thr56), the substrate of CaMK-III. About48 hours after siRNA transfection cells were treated with doxorubicinand lysed for Western blot analysis. (B) and (C) Inhibition of CaMK-IIIby siRNA led to reduced (B) p-Paxillin (Tyr-31), (C) phosphorylation ofmTOR and (D) VEGF in MDA-MB-231 cells. Cells were collected for Westernblot analysis 72 h after transfected with control siRNA or CaMK-IIIsiRNA.

FIG. 20. Analysis of MDA-MB-231 tumors (shown in FIG. 16A) samplesresected from mice after 4 weeks of treatment with liposomal-DOPC siRNA.(A) and (B). In vivo therapeutic targeting of CaMK-III by liposomalsiRNA decreased expression of Bcl-2 p-Src, p-FAK, VEGF also detected.(C) In vivo administration of L-CaMK-III siRNA inhibits target proteinprotein CaMK-III and phosphorylation of its downstream target EF2 inMDA-MBA231 tumors shown in FIG. 16B. The combination of liposomalCaMK-III siRNA treatment with doxorubicin enhanced inhibition of Bcl-2and HIF1α expression.

FIG. 21. Knockdown of CaMK-III by two different siRNA duplexes (#1 and#2[42]) enhances efficacy of doxorubicin (0.1 and 0.5 μM) inducedapoptosis of MDA-MB231 breast cancer cells. Apoptosis was detected byAnnexin V staining and the % of positive cells were quantified by FACSanalysis after 48 h of doxorubicin treatment.

FIG. 22. Inhibition of CaMK-III by siRNA enhances efficacy of paclitaxel(2 nM) inhibited colony formation of highly aggressive and metastaticMDA-MB231 breast cancer cells.

FIG. 23. Treatment schedule of liposomal siRNA (control and CaMK-III) innude mice bearing MDA-MB231 tumors. About 2 weeks after tumor cellinjection MDA-MB-231 cells, the targeting of the CaMK-III gene wasachieved by the systemic (i.v. from tail vein) administration ofliposomal siRNA (150 μg/kg or about 4 μg/mouse) twice a week for aperiod of 4 weeks. Treatment with two different siRNA duplexes (siRNA#1and siRNA#2[42]) targeting CaMK-III.

FIG. 24. To demonstrate that 5-HT plays a role in proliferation inbreast cancer cells we treated the triple negative (ER−, PR− andHer2/neu−) highly metastatic and aggressive breast cancer cell lineMDA-MB-231 with serotonin.

FIG. 25. 5-HT1B receptor is over expressed in breast cancer cell linesTo elucidate the mechanism of 5-HT-induced cell proliferation in breastcancer cells, 5-HTR1B receptor protein expression was assessed byWestern blot analysis in a normal breast epithelium MCF10A cell line andcompared to other breast cancer cell lines. These included the estrogenreceptor-negative human ER(−) breast cancer cell lines, MDA-MB-231,MDA-MB-435 and the estrogen receptor-positive ER(+) breast cancer celllines, MCF-7, chemotherapy (doxorubicin) resistant MCF-7/Dox-R cells,MCF10CA1a (CA1a), a metastatic human breast cancer line. All breastcancer cell lines showed higher expression of 5-HTR1B than the normalbreast epithelium MCF10A cell line.

FIG. 26. Specific inhibition of the 5-HT1B receptor inhibitsproliferation of breast cancer cells. To determine the role of the5-HT1B receptor we inhibited 5-HTR1B signaling in breast cancer cellsusing a specific antagonist SB224289 and assessed cell proliferation of[23]. This resulted in a dose-dependent inhibition of MCF7 (A), MB-231(B) and MCF7/Dox resistant (C) cell proliferation as detected by an MTSassay. Another specific antagonist (SB216641) of 5-HTR1B also showed asimilar ability to inhibit the growth of breast cancer cells. Theinhibition of 5-HT1B significantly inhibited the formation of MCF7 cellcolonies in a dose dependent manner (D). To study the effect of 5-HT1Bon the survival and colony formation of breast cancer cells a clonogenicsurvival assay was performed using various concentrations of SB224259over a period of two weeks.

FIG. 27. Inhibition of 5-HT1B leads to reduced colony formation ofbreast cancer cells. The appearance of cells after 72 h of treatment ofMDA-MB231 cells. Cell death and detachment as well as reduced cellnumber was observed. This assay is based on the ability of a single cellto grow into a colony [24]. Thus, compared to control experiments(untreated), SB224259-treated cells grew more slowly and produced fewercolonies. We found similar effects in MDA-MB-231 cells with SB224289.Overall, these data suggest that 5-HTR1B contributes to colony formationas inhibition of this receptor reduced colonies formed by breast cancercells.

FIG. 28. Inhibition of the 5-HT1B receptor induces autophagy andapoptosis in breast cancer cells. To determine the role of serotonin5-HT1B receptor in cell survival and the mechanism of growth inhibitioninduced by HT1B inhibitors we investigated cell death in breast cancercells. When 5-HT1B was inhibited by either SB224289 or SB216641 wedetected marked induction of autophagy in (A) as detected by acridineorange staining, apoptosis by Annexin V and necrosis by propidium iodide(PI) staining and FACS analysis (B) in MCF7 breast cancer cells.Induction of acidic vacuoles by acridine orange staining was alsoobserved in MDA-MB231 cells after SB224289 treatment for 72 hours(p<0.05) (FIG. 28C). Quantification of acridine orange stained vesicularorganelles by flow cytometry indicated that the percentage of positivecells in SB224289 or SB216641-treated cells (23% and 17%, respectively)were significantly higher than in the control untreated cells (2%).SB224289 treatment also induced apoptosis in a dose dependent manner inMCF cells (B). As the dose was increased SB224289 (higher than EC₅₀ 4.4μM) a shift from autophagy to apoptosis was observed, with reducedautophagy.

FIG. 29. Induction of autophagy was further evidenced by induction ofLC3-II, a specific marker of autophagy in response to SB224284 in MCF7and doxorubicin resistant MCF7 cells (MCF7/Dox-R) (A). To furtherdemonstrate that inhibition of HTR1B inhibition leads to autophagy inbreast cancer cells, we transfected MCF-7 cells with green fluorescentprotein-tagged LC3 (GFP-LC3) expression plasmid and determined theaccumulation of GFP-LC-II protein in autophagosomes after treatment withHTR1B inhibitor SB224289 (B). When autophagy is induced, LC3-II, acleaved product of LC3, specifically localizes to the membrane ofautophagosomes [17]. Therefore accumulation of GFP-LC3-II in thevacuoles following the SB224289 treatment indicates formation ofautophagosomes and induction of autophagy in the cells (B a-b). Incontrol or untreated cells, none of the above changes were observedexcept diffuse expression pattern of GFP-LC3 detected by fluorescencemicroscopy. In HTR1B inhibitor treated cells, GFP-LC3 distributes from adiffuse cytoplasmic pattern to form punctuate structures that indicatepreautophagosomal and autophagosomal membranes. (Bc-d) shows pictures ofthe cells with formation of autophagic vacuoles by phase contrastmicroscopy.

FIG. 30. To further support the findings we analyzed induction ofautophagy by transmission electron microscopy, which clearlydemonstrated that inhibition of HT1B by SB224289 or SB216641 treatmentinduced formation of autophagic vesicles containing cellular organelles,with merging of autophagic vesicles with lysosomes and lysed cellularcontent in the autophagosomes, indicating activity of lysosomal functionand degradation. These results suggest that inhibition of HTR1B inducesautophagy breast cancer cells.

FIG. 31. 5-HT1B modulates autophagy through activity of ERK1/2-MAPKsignaling in breast cancer cells. Induction of ERK1/2 has been shown toparticipate in the induction of autophagy. We investigated whetherinhibition of HT1B induces ERK-MAPK signaling in breast cancer cells.(A) and (B) Blockage of the 5-HT1B receptor with SB224289 inducedphosphorylation of ERK (p42/p44) in MCF7, MCF7/Dox resistant andMDA-MB-231 as detected by Western blot analysis (FIG. 31-32).

FIG. 32. Induction of ERK1/2 has been shown to participate in theinduction of autophagy. We investigated whether inhibition of HT1Binduces ERK-MAPK signaling in breast cancer cells. Blockage of the5-HT1B receptor with SB224289 induced phosphorylation of ERK (p42/p44)in MCF7, MCF7/Dox resistant and MDA-MB-231 as detected by Western blotanalysis (FIG. 31-32). SB216641 also induced phosphorylation of ERK(p42/p44) and LC3-III in MCF7 cells (A). Induction of ERK signaling wasclosely associated with induction of autophagy marker LC3-II in alltreatments and breast cancer cell lines tested including MDA-MB231 inresponse to inhibition of HT1B signaling by 2 different inhibitors (B).

FIG. 33. To demonstrate a link between autophagy induction in responseto HTR1B inhibition and ERK activation we inhibited ERK using a MEKinhibitor (PD98059) we transfected MCF-7 cells with green fluorescentprotein-tagged LC3 (GFP-LC3) expression plasmid and determined theaccumulation of GFP-LC-II protein in autophagosomes after treatment withHTR1B inhibitor SB224289 (A) and (B). Accumulation of GFP-LC3-II in thevacuoles following pretreatment with PD98059 prevented SB224289 inducedformation of autophagosomes and punctuate pattern indicating inductionof autophagy in the cells (FIG. 29B a-b) detected by fluorescencemicroscopy. In HTR1B inhibitor treated cells, GFP-LC3 showed punctuatestructures that indicate preautophagosomal and autophagosomal membranes.(B) shows number of GFP-LC3 positive cells formation of autophagicvacuoles with or without the treatment of ERK inhibitor.

FIG. 34. To further demonstrate that autophagy induced in response toHTR1B inhibition is mediated by ERK activation we inhibited ERK using aMEK inhibitor (PD98059) and found that inhibition of ERK significantlyreduced autophagy induction in MCF7 and MDA-MB231 cells detected byacridine orange staining and FACS analysis (p<0.05) (A) and (B). Thepercent positive cells with autophagy induced by SB224359 was decreasedto 18% from 29.2% by ERK inhibitor, suggesting that ERK mediatesparticipates to autophagy induced by inhibition of HTR1B.

FIG. 35. Induction of autophagy by HT1B inhibition contributes to celldeath. Autophagy has been shown to function as a protective survivalpathway in response to nutrient or growth factor deprivation, hypoxia,metabolic and therapeutic stress [25]. It was also reported thatautophagy induction can lead to cells death [16][19]. To understand therole of autophagy in response to HT1B inhibition by SB224289 weinhibited autophagy by 3-MA (A). The incidence of autophagy induced bySB224359 was decreased to 12% from 22.4% by 3-MA. Cell viability wassignificantly increased by 3-MA from 42% by SB224289 to 61% and 31% bySB216641 to 71% in MCF7 cells (p<0.05) (B). These results suggest thatinhibition of autophagy increases cell viability by HTR1B inhibition andautophagy contributes to cell death.

FIG. 36. Cell viability MDA-231 cells.

FIG. 37. 5-NT and KD06 tested in a large panel of breast cancer cells.Both compounds are effective against almost all breast cancer celllines, including: Triple negative (estrogen receptor negative; ER(−),progesterone recetor negative and Her2/neu negative) breast cancer celllines) including MDA-468,BT20 and MDa-MB231 cells. Triple negativebreast cancer patients have poor prognosis and they have resistance tostandard therapies and cannot use tamoxifen antiestrogen therapy orherception antibody as targeted therapy since they do not have targetsfor these therapies. SKBR3, MDA-MB435, T47D, ZR75.1 breast cancer celllines with different features, such as high SRC, HER2 etc. are sensitiveto 5-NT and KD06. Y-axes for all views shown in this figure are cellsurvival (O.D.).

FIG. 38. (A). 5-NT and KD06 at IC50 to IC90 doses in breast cancer cells(MCF7) are not toxic against normal breast cancer epithelium (MCF10A).(B). Animals(mice) that received at least 15 doses of liposomeincorporated 5-NT (i.v, 10 mg/kg) for 4 weeks look Ok compared tolipsome incorporated control siRNA treated group. (C). Liver (AST, ALT),kidney (BUN, Creatinine) tox markers. Mice that received 5-NT for 4weeks did not lose any weight and they looked healthy (AST:Aspartatetransaminase, ALT:Alanine transaminase, ALP:Alkaline phosphatase,BUN:blood urea nitrogen) (Each grouping is shown left to right AST, ALT,ALP, BUN, creatinine).

FIG. 39. Inhibition of HTR1B/1D by siRNA mediated gene silencingreverses some of the effect of 5NT and KD06 on cell proliferationinhibition, suggesting that 5NT inhibits cell proliferation through HTR1B/1D receptors.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl,homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds. Examples of unsaturated alkyl groupsinclude, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. An alkoxy is an alkyl attached to the remainder of the moleculevia an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms. Theterm “alkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom selected from the group consisting of O, N, P, Si, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized,and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′- and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,and the like. Examples of heterocycloalkyl include, but are not limitedto, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. An “arylene” and a “heteroarylene,” alone or as part ofanother substituent, mean a divalent radical derived from an aryl andheteroaryl, respectively. Non-limiting examples of heteroaryl groupsinclude pyridinyl, pyrimidinyl, thiophenyl, furanyl, indolyl,benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl,pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl,quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl,benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl,imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl,pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl,isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl,tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl,pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. Theexamples above may be substituted or unsubstituted and divalent radicalsof each heteroaryl example above are non-limiting examples ofheteroarylene.

A fused ring heterocyloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substitutentsdescribed herein.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R′ is a substituted or unsubstituted alkylgroup as defined above. R′ may have a specified number of carbons (e.g.,“C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R″″, —CN, —NO₂, in a number ranging from zeroto (2m′+1), where m′ is the total number of carbon atoms in suchradical. R, R′, R″, R′″, and R″″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted heteroaryl, substituted orunsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″, and R″″ group when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 4-, 5-, 6-, or 7-memberedring. For example, —NR′R″ includes, but is not limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C═(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system; and where R′, R″,R′″, and R″″ are preferably independently selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″, and R″″ groupswhen more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, —COOH,        —CONH₂, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, unsubstituted alkyl, unsubstituted heteroalkyl,        unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,        unsubstituted aryl, unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, —COOH,            —CONH₂, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,            —NHC═(O)NHNH₂, unsubstituted alkyl, unsubstituted            heteroalkyl, unsubstituted cycloalkyl, unsubstituted            heterocycloalkyl, unsubstituted aryl, unsubstituted            heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            and heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,                —COOH, —CONH₂, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,                —ONH₂, —NHC═(O)NHNH₂, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, or heteroaryl, substituted with at least one                substituent selected from: oxo, —OH, —NH₂, —SH, —CN,                —CF₃, —NO₂, halogen, —COOH, —CONH₂, —SO₂Cl, —SO₃H,                —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, and unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section below.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853). Themethods above may be used to synthesize single molecular species.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different.

Description of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The terms “treating” or “treatment” refers to any indicia of success inthe treatment or amelioration of an injury, disease, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,the certain methods presented herein successfully treat cancer bydecreasing the incidence of cancer and or causing remission of cancer.The term “treating,” and conjugations thereof, include prevention of aninjury, pathology, condition, or disease.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease, reduce enzyme activity, increase enzyme activity, reduce one ormore symptoms of a disease or condition, reduce the level of a kinaseactivity in a cell, reduce the level of activity of eEF-2K, reduce thelevel of activity of a serotonin receptor in a cell, reduce the level ofactivity of a 5-HTR_(1B) in a cell, reduce the level of activity of a5-HTR_(1D) in a cell, reduce the level of activity of mTOR, c-myc,cyclin-D1. Bcl-2, VEGF, HIF1alpha, c-SRC, Fak, Paxillin, IGR-1R, and/orAKT in a cell, increase the level of activity of a serotonin receptor ina cell, increase the level of activity of a 5-HTR_(1B) in a cell, orincrease the level of activity of a 5-HTR_(1D) in a cell). An example ofan “effective amount” is an amount sufficient to contribute to thetreatment, prevention, or reduction of a symptom or symptoms of adisease, which could also be referred to as a “therapeutically effectiveamount.” A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). A“prophylactically effective amount” of a drug is an amount of a drugthat, when administered to a subject, will have the intendedprophylactic effect, e.g., preventing or delaying the onset (orreoccurrence) of an injury, disease, pathology or condition, or reducingthe likelihood of the onset (or reoccurrence) of an injury, disease,pathology, or condition, or their symptoms. The full prophylactic effectdoes not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a prophylacticallyeffective amount may be administered in one or more administrations. An“activity decreasing amount,” as used herein, refers to an amount ofantagonist (inhibitor) required to decrease the activity of an enzymerelative to the absence of the antagonist. An “activity increasingamount,” as used herein, refers to an amount of agonist (activator)required to increase the activity of an enzyme relative to the absenceof the agonist. A “function disrupting amount,” as used herein, refersto the amount of antagonist (inhibitor) required to disrupt the functionof an enzyme or protein relative to the absence of the antagonist. A“function increasing amount,” as used herein, refers to the amount ofagonist (activator) required to increase the function of an enzyme orprotein relative to the absence of the agonist. The exact amounts willdepend on the purpose of the treatment, and will be ascertainable by oneskilled in the art using known techniques (see, e.g., Lieberman,Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Scienceand Technology of Pharmaceutical Compounding (1999); Pickar, DosageCalculations (1999); and Remington: The Science and Practice ofPharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams &Wilkins).

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules, or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be a compoundas described herein and a protein or enzyme (e.g. kinase, eEF-2K,receptor, 5-HTR_(1B), or 5-HTR_(1D)). In some embodiments, the proteinmay be a 5-HTR. In some embodiments, the protein may be 5-HTR_(1B). Insome embodiments, the protein may be 5-HTR_(1D). In some embodiments,the protein may be eEF-2K. In some embodiments contacting includesallowing a compound described herein to interact with a protein orenzyme that is involved in a signaling pathway.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor (e.g. antagonist)interaction means negatively affecting (e.g. decreasing) the activity orfunction of the protein (e.g. decreasing the phosphorylation of anotherprotein by a kinase) relative to the activity or function of the protein(e g kinase) in the absence of the inhibitor (e.g. kinase inhibitor orkinase inhibitor compound). In some embodiments inhibition refers toreduction of a disease or symptoms of disease. In some embodiments,inhibition refers to a reduction in the activity of a signaltransduction pathway or signaling pathway (e.g. reduction of a pathwayinvolving a 5-HTR, 5-HTR_(1B), 5-HTR_(1D), or eEF-2K). Thus, inhibitionincludes, at least in part, partially or totally blocking stimulation,decreasing, preventing, or delaying activation, or inactivating,desensitizing, or down-regulating signal transduction or enzymaticactivity or the amount of a protein (e.g. a 5-HTR, 5-HTR_(1B),5-HTR_(1D), eEF-2K, or phosphorylated eEF-2). In some embodiments,inhibition refers to inhibition of a protein, such as 5-HTR_(1B),5-HTR_(1D), eEF-2K, or eEF-2. In some embodiments, the 5-HTR,5-HTR_(1B), 5-HTR_(1D), eEF-2K, or phosphorylated eEF-2 is a humanprotein.

As defined herein, the term “activation”, “activate”, “activating” andthe like in reference to a protein-activator (e.g. agonist) interactionmeans positively affecting (e.g. increasing) the activity or function ofthe protein (e.g. increasing the phosphorylation of another protein by akinase) relative to the activity or function of the protein (e g kinase)in the absence of the activator (e g kinase activator or kinaseactivator compound or kinase agonist, 5-HTR agonist or activator,5-HTR_(1B) agonist or activator, or 5-HTR_(1D) agonist or activator). Insome embodiments, activation refers to an increase in the activity of asignal transduction pathway or signaling pathway (e.g. activation of apathway involving a 5-HTR, 5-HTR_(1B), 5-HTR_(1D), or eEF-2K). Thus,activation includes, at least in part, partially or totally increasingstimulation, increasing or enabling activation, or activating,sensitizing, or up-regulating signal transduction or enzymatic activityor the amount of a protein (e.g. a 5-HTR, 5-HTR_(1B), 5-HTR_(1D),eEF-2K, or de-phosphorylated eEF-2). In some embodiments, activationrefers to activation of a protein, such as 5-HTR_(1B), 5-HTR_(1D),eEF-2K, or eEF-2. In some embodiments, the 5-HTR, 5-HTR_(1B),5-HTR_(1D), eEF-2K, or de-phosphorylated eEF-2 is a human protein.

The term “modulator” refers to a composition that increases or decreasesthe level of a target molecule or the function of a target molecule(e.g. a target may be a kinase (e.g. eEF-2K) and the function may be tophosphorylate a molecule (e.g. eEF-2) or the target may be a 5-HTR andthe function may be the downstream signaling pathway or phosphorylationof eEF-2 or the target may be eEF-2 and the function may be theribosomal translocation of the nascent peptide chain from the A-site tothe P-site during translation). In some embodiments, a kinase modulatoris a compound that reduces the activity of a kinase in a cell. A kinasemodulator may reduce the activity of one kinase but cause an increase inenzyme activity of another kinase that results in a reduction orincrease, respectively, of cell growth and proliferation. In someembodiments, a kinase disease modulator is a compound that reduces theseverity of one or more symptoms of a disease associated with the kinase(e.g. cancer). A 5-HTR modulator is a compound that increases ordecreases the activity or level of one or more 5-HTRs. A 5-HTR_(1B)modulator is a compound that increases or decreases the activity orlevel of 5-HTR_(1B). A 5-HTR_(1D) modulator is a compound that increasesor decreases the activity or level of 5-HTR_(1D). An eEF-2K modulator isa compound that increases or decreases the activity or level of eEF-2K.An eEF-2 modulator is a compound that increases or decreases theactivity or level of eEF-2K.

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a compound, inhibitory nucleic acid, or pharmaceuticalcomposition, all as provided herein. Non-limiting examples includehumans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep,cows, deer, and other non-mammalian animals. In some embodiments, apatient is human.

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with a compound, inhibitorynucleic acid, pharmaceutical composition, or method provided herein. Insome embodiments, the disease is a disease related to (e.g. caused by)an activated or overactive kinase or aberrant kinase activity (e.g.cancer with increased level of eEF-2K activity or increased signaltransduction activity in pathways involving 5-HTR_(1B) or 5-HTR_(1D)).In some embodiments, the disease is a disease related to (e.g. causedby) an inhibited kinase or reduced kinase activity (e.g. cancer withdecreased level of eEF-2 activity or decreased signal transductionactivity in pathways involving 5-HTR_(1B) or 5-HTR_(1D)). Examples ofdiseases, disorders, or conditions include, but are not limited to,cancer, melanoma, breast cancer, ovarian cancer, pancreatic cancer,liver cancer, metastatic cancer, lung cancer, glioblastoma, glioma,prostate cancer, leukemia, sarcoma, carcinoma, lymphoma, neuroblastoma,depression, major depression, chronic depression, atypical depression,bipolar depression, seasonal depression, anxiety, compulsive behavior,addiction, post-traumatic stress syndrome, major psychotic depression,stress disorders, cognitive impairment in depressed patients, chronicpain, postpartum psychosis, postpartum depression, neurologicaldisorders in premature infants, migraine headaches, or psychoticdepression. In some instances, “disease” or “condition” refer to cancer.In some further instances, “cancer” refers to human cancers andcarcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, melanomas,etc., including solid and lymphoid cancers, kidney, breast, lung,bladder, colon, ovarian, prostate, pancreas, stomach, brain, head andneck, skin, uterine, testicular, glioma, esophagus, and liver cancer,including hepatocarcinoma, lymphoma, including B-acute lymphoblasticlymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, andLarge Cell lymphomas), Hodgkin's lymphoma, leukemia (including AML, ALL,and CML), or multiple myeloma.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemia,carcinomas and sarcomas. Exemplary cancers that may be treated with acompound, inhibitory nucleic acid, pharmaceutical composition, or methodprovided herein include breast cancer (e.g. ER positive, ER negative,chemotherapy resistant, herceptin resistant, HER2 positive, doxorubicinresistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma,primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer(e.g. hepatocellular carcinoma), lung cancer (e.g. non-small cell lungcarcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lungcarcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastomamultiforme, glioma, or melanoma. Additional examples include, cancer ofthe thyroid, endocrine system, brain, breast, cervix, colon, head &neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma,ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma,glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, primary brain tumors, cancer,malignant pancreatic insulanoma, malignant carcinoid, urinary bladdercancer, premalignant skin lesions, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, endometrial cancer, adrenal cortical cancer,neoplasms of the endocrine or exocrine pancreas, medullary thyroidcancer, medullary thyroid carcinoma, melanoma, colorectal cancer,papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease ofthe Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma,cancer of the pancreatic stellate cells, cancer of the hepatic stellatecells, or prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). Exemplary leukemias that may be treated with a compound,inhibitory nucleic acid, pharmaceutical composition, or method providedherein include, for example, acute nonlymphocytic leukemia, chroniclymphocytic leukemia, acute granulocytic leukemia, chronic granulocyticleukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemicleukemia, a leukocythemic leukemia, basophylic leukemia, blast cellleukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis,embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cellleukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocyticleukemia, stem cell leukemia, acute monocytic leukemia, leukopenicleukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocyticleukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cellleukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblasticleukemia, monocytic leukemia, myeloblastic leukemia, myelocyticleukemia, myeloid granulocytic leukemia, myelomonocytic leukemia,Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacyticleukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling'sleukemia, stem cell leukemia, subleukemic leukemia, or undifferentiatedcell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound, inhibitorynucleic acid, pharmaceutical composition, or method provided hereininclude a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma,myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma,liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoidsarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms'tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma,fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocyticsarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagicsarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblasticsarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cellsarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma,parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocysticsarcoma, synovial sarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a compound, inhibitory nucleic acid, pharmaceuticalcomposition, or method provided herein include, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma, subungal melanoma, or superficial spreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound, inhibitory nucleic acid, pharmaceutical composition, or methodprovided herein include, for example, medullary thyroid carcinoma,familial medullary thyroid carcinoma, acinar carcinoma, acinouscarcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinomaadenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolarcell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, ductal carcinoma, carcinoma durum, embryonalcarcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinomaepitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere,carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giantcell carcinoma, carcinoma gigantocellulare, glandular carcinoma,granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

A “cancer associated with aberrant 5-HTR_(1B) activity” (also referredto herein as “5-HTR_(1B) related cancer”) is a cancer caused by aberrant5-HTR_(1B) activity (e.g. increased or decreased level of activity of5-HTR_(1B)). A “cancer associated with aberrant 5-HTR_(1D) activity”(also referred to herein as “5-HTR_(1D) related cancer”) is a cancercaused by aberrant 5-HTR_(1D) activity (e.g. an increased or decreasedlevel of activity of 5-HTR_(1D)). A “cancer associated with aberranteEF-2K activity” (also referred to herein as “eEF-2K related cancer”) isa cancer caused by aberrant eEF-2K activity (e.g. a mutated eEF-2K geneor abnormal (increased or decreased) level of activity of eEF-2K).

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “preparation” is intended to include the formulation of theactive compound or inhibitory nucleic acid with encapsulating materialas a carrier providing a capsule in which the active component with orwithout other carriers, is surrounded by a carrier, which is thus inassociation with it. Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intrathecal, intranasalor subcutaneous administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to a subject. Administration is byany route, including parenteral and transmucosal (e.g., buccal,sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).Parenteral administration includes, e.g., intravenous, intramuscular,intra-arteriole, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial. Other modes of delivery include, butare not limited to, the use of liposomal formulations, intravenousinfusion, transdermal patches, etc. By “co-administer” it is meant thata composition described herein is administered at the same time, justprior to, or just after the administration of one or more additionaltherapies, for example cancer therapies such as chemotherapy, hormonaltherapy, radiotherapy, or immunotherapy. The compound or inhibitorynucleic acid of the invention can be administered alone or can becoadministered to the patient. Coadministration is meant to includesimultaneous or sequential administration of the compound or inhibitorynucleic acid individually or in combination (more than one compound orinhibitory nucleic acid). Thus, the preparations can also be combined,when desired, with other active substances (e.g. to reduce metabolicdegradation). The compositions of the present invention can be deliveredby transdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

The term “administer (or administering) a 5-HTR inhibitor” meansadministering a compound or inhibitory nucleic acid that inhibits theactivity or reduces the level (e.g. amount) of one or more 5-HTR(s)(e.g. a 5-HTR_(1B) inhibitor, HTR_(1D) inhibitor, HTR_(1B/1D) inhibitor,a pan 5-HTR inhibitor) to a subject and, without being limited bymechanism, allowing sufficient time for the 5-HTR inhibitor to reducethe activity of one or more 5-HTR(s), for the 5-HTR inhibitor to reducethe level (e.g. amount) of one or more 5-HTR(s), or for the 5-HTRinhibitor to reduce one or more symptoms of a disease (e.g. cancer,wherein the 5-HTR inhibitor may arrest the cell cycle, slow the cellcycle, reduce DNA replication, reduce cell replication, reduce cellgrowth, reduce metastasis, reduce proliferation of cancer cells, reducemigration, reduce angiogenesis, or cause cell death).

The term “administer (or administering) a 5-HTR activator” meansadministering a compound or inhibitory nucleic acid that increases theactivity or level (e.g. amount) of one or more 5-HTR(s) (e.g. a5-HTR_(1B) activator, HTR_(1D) activator, HTR_(1B/1D) activator, a pan5-HTR activator) to a subject and, without being limited by mechanism,allowing sufficient time for the 5-HTR activator to increase theactivity of one or more 5-HTR(s), to increase the level (e.g. amount) ofone or more 5-HTR(s), or for the 5-HTR activator to reduce one or moresymptoms of a disease (e.g. cancer, wherein the 5-HTR activator mayarrest the cell cycle, slow the cell cycle, reduce DNA replication,reduce cell replication, reduce cell growth, reduce metastasis, reduceproliferation of cancer cells, reduce migration, reduce angiogenesis, orcause cell death).

The term “administer (or administering) an eEF-2K inhibitor” meansadministering a compound or inhibitory nucleic acid that inhibits theactivity or reduces the level (e.g. amount) of eEF-2K to a subject and,without being limited by mechanism, allowing sufficient time for theeEF-2K inhibitor to reduce the activity of eEF-2K, for the eEF-2Kinhibitor to reduce the level (e.g. amount) of eEF-2K, or for the eEF-2Kinhibitor to reduce one or more symptoms of a disease (e.g. cancer,wherein the eEF-2K inhibitor may arrest the cell cycle, slow the cellcycle, reduce DNA replication, reduce cell replication, reduce cellgrowth, reduce metastasis, reduce proliferation of cancer cells, reducemigration, reduce angiogenesis, or cause cell death).

The term “associated” or “associated with” as used herein to describe adisease (e.g. a protein associated disease, a cancer associated withaberrant 5-HTR activity, a cancer associated with aberrant 5-HTR_(1B)activity, a cancer associated with aberrant 5-HTR_(1D) activity, acancer associated with aberrant eEF-2K activity, 5-HTR_(1B) associatedcancer, 5-HTR_(1D) associated cancer, 5-HTR associated cancer, eEF-2Kassociated cancer, depression or migraine pain or pain associated with5-HTR activity, 5-HTR_(1B) activity, 5-HTR_(1D) activity, or eEF-2Kactivity) means that the disease (e.g. cancer, depression, migrainepain, pain) is caused by, or a symptom of the disease is caused by, whatis described as disease associated or what is described as associatedwith the disease. For example, a cancer associated with aberrant5-HTR_(1B) activity may be a cancer that results (entirely or partially)from aberrant 5-HTR_(1B) activity or a cancer wherein a particularsymptom of the disease is caused (entirely or partially) by aberrant5-HTR_(1B) activity. As used herein, what is described as beingassociated with a disease, if a causative agent, could be a target fortreatment of the disease. For example, a cancer associated with aberrant5-HTR_(1B) activity or a 5-HTR_(1B) associated cancer, may be treatedwith a 5-HTR_(1B) modulator or 5-HTR_(1B) inhibitor, in the instancewhere increased 5-HTR_(1B) activity causes the cancer or 5-HTR_(1B)activator, in the instance where decreased 5-HTR_(1B) activity causesthe cancer.

The tem “aberrant” as used herein refers to different from normal. Whenused to described enzymatic or signal transduction activity, aberrantrefers to activity that is greater or less than a normal control or theaverage of normal non-diseased control samples. Aberrant activity mayrefer to an amount of activity that results in a disease, whereinreturning the aberrant activity to a normal or non-disease-associatedamount (e.g. by administering a compound or inhibitory nucleic acid orusing a method as described herein), results in reduction of the diseaseor one or more disease symptoms.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. The term “nucleic acid” includes single-, double-, ormultiple-stranded DNA, RNA and analogs (derivatives) thereof.Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25,30, 40, 50 or more nucleotides in length, up to about 100 nucleotides inlength. Nucleic acids and polynucleotides are a polymers of any length,including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000,7000, 10,000, etc. In certain embodiments. the nucleic acids hereincontain phosphodiester bonds. In other embodiments, nucleic acid analogsare included that may have alternate backbones, comprising, e.g.,phosphoramidate, phosphorothioate, phosphorodithioate, orO-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press); and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made.

A particular nucleic acid sequence also encompasses “splice variants.”Similarly, a particular protein encoded by a nucleic acid encompassesany protein encoded by a splice variant of that nucleic acid. “Splicevariants,” as the name suggests, are products of alternative splicing ofa gene. After transcription, an initial nucleic acid transcript may bespliced such that different (alternate) nucleic acid splice productsencode different polypeptides. Mechanisms for the production of splicevariants vary, but include alternate splicing of exons. Alternatepolypeptides derived from the same nucleic acid by read-throughtranscription are also encompassed by this definition. Any products of asplicing reaction, including recombinant forms of the splice products,are included in this definition.

The terms “identical” or percent sequence “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site at ncbi.nlm.nih.gov/BLAST/ or the like). Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the compliment of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. Employed algorithmscan account for gaps and the like.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 10 to 600, usually about 20 to about 200, moreusually about 50 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence with a higher affinity, e.g., under more stringentconditions, than to other nucleotide sequences (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent hybridization conditions areselected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent hybridization conditions may also be achievedwith the addition of destabilizing agents such as formamide. Forselective or specific hybridization, a positive signal is at least twotimes background, preferably 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley& Sons.

Nucleic acids may be substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.

An “inhibitory nucleic acid” is a nucleic acid (e.g. DNA, RNA, polymerof nucleotide analogs) that is capable of binding to a target nucleicacid (e.g. an mRNA translatable into an eEF-2K, 5-HTR, 5-HTR_(1B), or5-HTR_(1D)) and reducing transcription of the target nucleic acid (e.g.mRNA from DNA) or reducing the translation of the target nucleic acid(e.g. mRNA) or altering transcript splicing. In some embodiments, the“inhibitory nucleic acid” is a nucleic acid that is capable of binding(e.g. hybridizing) to a target nucleic acid (e.g. an mRNA translatableinto an eEF-2K, 5-HTR, 5-HTR_(1B), or 5-HTR_(1D)) and reducingtranslation of the target nucleic acid. The target nucleic acid is orincludes one or more target nucleic acid sequences to which theinhibitory nucleic acid binds (e.g. hybridizes). Thus, an inhibitorynucleic acid typically is or includes a sequence (also referred toherein as an “antisense nucleic acid sequence”) that is capable ofhybridizing to at least a portion of a target nucleic acid at a targetnucleic acid sequence. An example of an inhibitory nucleic acid is anantisense nucleic acid. Another example of an inhibitory nucleic acid issiRNA or RNAi (including their derivatives or pre-cursors, such asnucleotide analogs). Further examples include shRNA, miRNA, shmiRNA, orcertain of their derivatives or pre-cursors. In some embodiments, theinhibitory nucleic acid is single stranded. In other embodiments, theinhibitory nucleic acid is double stranded. An “anti-eEF-2K inhibitorynucleic acid” is an inhibitory nucleic acid that is capable of bindingto a nucleic acid that codes for at least a portion of eEF-2K andreducing transcription or translation of the target nucleic acid oraltering transcript splicing. An “anti-5-HTR_(1B) inhibitory nucleicacid” is an inhibitory nucleic acid that is capable of binding to anucleic acid that codes for at least a portion of 5-HTR_(1B) andreducing transcription or translation of the target nucleic acid oraltering transcript splicing. An “anti-5-HTR_(1D) inhibitory nucleicacid” is an inhibitory nucleic acid that is capable of binding to anucleic acid that codes for at least a portion of 5-HTR_(1D) andreducing transcription or translation of the target nucleic acid oraltering transcript splicing. An “anti-5-HTR inhibitory nucleic acid” isan inhibitory nucleic acid that is capable of binding to a nucleic acidthat codes for at least a portion of a 5-HTR and reducing transcriptionor translation of the target nucleic acid or altering transcriptsplicing.

An “antisense nucleic acid” is a nucleic acid (e.g. DNA, RNA or analogsthereof) that is at least partially complementary to at least a portionof a specific target nucleic acid (e.g. a target nucleic acid sequence),such as an mRNA molecule (e.g. a target mRNA molecule) (see, e.g.,Weintraub, Scientific American, 262:40 (1990)), for example antisense,siRNA, shRNA, shmiRNA, miRNA (microRNA). Thus, antisense nucleic acidsare capable of hybridizing to (e.g. selectively hybridizing to) a targetnucleic acid (e.g. target mRNA). In some embodiments, the antisensenucleic acid hybridizes to the target nucleic acid sequence (e.g. mRNA)under stringent hybridization conditions. In some embodiments, theantisense nucleic acid hybridizes to the target nucleic acid (e.g. mRNA)under moderately stringent hybridization conditions. Antisense nucleicacids may comprise naturally occurring nucleotides or modifiednucleotides such as, e.g., phosphorothioate, methylphosphonate, and-anomeric sugar-phosphate, backbone-modified nucleotides. An“anti-eEF-2K antisense nucleic acid” is an antisense nucleic acid thatis at least partially complementary to at least a portion of a targetnucleic acid sequence, such as an mRNA molecule, that codes for at leasta portion of eEF-2K. An “anti-5-HTR_(1B) antisense nucleic acid” is anantisense nucleic acid that is at least partially complementary to atleast a portion of a target nucleic acid sequence, such as an mRNAmolecule, that codes for at least a portion of the 5-HTR_(1B). An“anti-5-HTR_(1D) antisense nucleic acid” is an antisense nucleic acidthat is at least partially complementary to at least a portion of atarget nucleic acid sequence, such as an mRNA molecule, that codes forat least a portion of the 5-HTR_(1D). An “anti-5-HTR antisense nucleicacid” is an antisense nucleic acid that is at least partiallycomplementary to at least a portion of a target nucleic acid sequence,such as an mRNA molecule, that codes for at least a portion of a 5-HTR.Antisense nucleic acids may be single or double stranded nucleic acids.

In the cell, the antisense nucleic acids may hybridize to the targetmRNA, forming a double-stranded molecule. The antisense nucleic acids,interfere with the translation of the mRNA, since the cell will nottranslate a mRNA that is double-stranded. The use of antisense methodsto inhibit the in vitro translation of genes is well known in the art(Marcus-Sakura, Anal. Biochem., 172:289, (1988)). Antisense moleculeswhich bind directly to the DNA may be used.

Inhibitory nucleic acids can be delivered to the subject using anyappropriate means known in the art, including by injection, inhalation,or oral ingestion. Another suitable delivery system is a colloidaldispersion system such as, for example, macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexample of a colloidal system of this invention is a liposome (e.g.dioleoyl-sn-glycero-3-phosphocholine containing ordimyristoyl-phosphatidylcholine containing) Liposomes are artificialmembrane vesicles which are useful as delivery vehicles in vitro and invivo. Nucleic acids, including RNA and DNA within liposomes can bedelivered to cells in a biologically active form (Fraley, et al., TrendsBiochem. Sci., 6:77, 1981). The compounds described herein (e.g. Formula(I), (II), (III), or (IV), including embodiments) may also be deliveredto cells by liposomes. Liposomes can be targeted to specific cell typesor tissues using any means known in the art (e.g. by modifying theliposome with RGD or folate). Inhibitory nucleic acids (e.g. antisensenucleic acids, siRNAs) may be delivered to a cell using cell permeabledelivery systems (e.g. cell permeable peptides). In some embodiments,inhibitory nucleic acids are delivered to specific cells or tissuesusing viral vectors or viruses.

An “siRNA” refers to a nucleic acid that forms a double stranded RNA,which double stranded RNA has the ability to reduce or inhibitexpression of a gene or target gene when the siRNA is present (e.g.expressed) in the same cell as the gene or target gene. The siRNA istypically about 5 to about 100 nucleotides in length, more typicallyabout 10 to about 50 nucleotides in length, more typically about 15 toabout 30 nucleotides in length, most typically about 20-30 basenucleotides, or about 20-25 or about 24-29 nucleotides in length, e.g.,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.siRNA molecules and methods of generating them are described in, e.g.,Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411,494-498; WO 00/44895; WO 01/36646; WO 99/32619; WO 00/01846; WO01/29058; WO 99/07409; and WO 00/44914. A DNA molecule that transcribesdsRNA or siRNA (for instance, as a hairpin duplex) also provides RNAi.DNA molecules for transcribing dsRNA are disclosed in U.S. Pat. No.6,573,099, and in U.S. Patent Application Publication Nos. 2002/0160393and 2003/0027783, and Tuschl and Borkhardt, Molecular Interventions,2:158 (2002). An “anti-eEF-2K siRNA” is an siRNA that is at leastpartially complementary to at least a portion of a target nucleic acidsequence, such as an mRNA molecule, that codes for at least a portion ofeEF-2K. An “anti-5-HTR_(1B) siRNA” is an siRNA that is at leastpartially complementary to at least a portion of a target nucleic acidsequence, such as an mRNA molecule, that codes for at least a portion ofthe 5-HTR_(1B). An “anti-5-HTR_(1D) siRNA” is an siRNA that is at leastpartially complementary to at least a portion of a target nucleic acidsequence, such as an mRNA molecule, that codes for at least a portion ofthe 5-HTR_(1D). An “anti-5-HTR siRNA” is an siRNA that is at leastpartially complementary to at least a portion of a target nucleic acidsequence, such as an mRNA molecule, that codes for at least a portion ofa 5-HTR.

The siRNA can be administered directly or siRNA expression vectors canbe used to induce RNAi that have different design criteria. A vector canhave inserted two inverted repeats separated by a short spacer sequenceand ending with a string of T's which serve to terminate transcription.

Construction of suitable vectors containing the desired therapeutic genecoding and control sequences employs standard ligation and restrictiontechniques, which are well understood in the art (see Maniatis et al.,in Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1982)). Isolated plasmids, DNA sequences, orsynthesized oligonucleotides are cleaved, tailored, and re-ligated inthe form desired.

The term “eEF-2” refers to the eukaryotic (translation) elongationfactor 2 (e.g. Homo sapien accession no. NM_(—)0001961). eEF-2 may referto the protein, DNA, or mRNA.

The term “eEF-2K” refers to the eukaryotic elongation factor 2 kinase(e.g. Homo sapien accession no. NM_(—)013302). eEF-2 may refer to theprotein, DNA, or mRNA. eEF-2K is also called “CaMK-III” and is alsoknown as Calmodulin-dependent protein kinase-III, and these terms may beused interchangeably.

The term “5-HTR_(1B)” refers to the 5-hydroxytryptamine (serotonin)receptor 1B (e.g. Homo sapien accession no. NM_(—)000863). 5-HTR_(1B)may refer to the protein, DNA, or mRNA.

The term “5-HTR_(1D)” refers to the 5-hydroxytryptamine (serotonin)receptor 1D (e.g. Homo sapien accession no. NM_(—)000864). 5-HTR_(1D)may refer to the protein, DNA, or mRNA.

The term “5-HTR” refers to a 5-hydroxytryptamine (serotonin) receptor(e.g. selected from 5-HTR₁ through 5-HTR, including subtypes). 5-HTR mayrefer to the protein, DNA, or mRNA.

Compounds

In a first aspect, a compound is provided having the formula:

R^(1A) is independently hydrogen, halogen, —CX^(1A) ₃, —C(O)R^(7A),—C(O)—OR^(7A), —C(O)NR^(7A)R^(8A), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted memberedheteroaryl. R^(1B) is independently hydrogen, halogen, —CX^(1B) ₃,—C(O)R^(7B), —C(O)—OR^(7B), —C(O)NR^(7B)R^(8B), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R^(2A) is independently hydrogen, halogen,—CX^(2A) ₃, —C(O)R^(9A), —C(O)—OR^(9A), —C(O)NR^(9A)R^(10A), substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R^(2B) is independently hydrogen, halogen,—CX^(2B) ₃, —C(O)R^(9B), —C(O)—OR^(9B), —C(O)NR^(9B), R^(10B),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R^(1A) and R^(2A) areoptionally joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. R^(1B) andR^(2B) are optionally joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. R³ isindependently hydrogen, halogen, —CX³ ₃, —CN, —SO₂Cl, —SO_(n)R¹⁴,—SO_(k)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m), —NR¹¹R¹², —C(O)R¹³, —C(O)—OR¹³, —O—C(O)—R¹³, —C(O)NR¹¹R¹²,—NR¹¹C(O)R¹³, —OR¹⁴, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R^(7A),R^(7B), R^(8A), R^(8B), R^(9A), R^(9B), R^(10A), R^(10B), R¹¹, R¹², R¹³,and R¹⁴ are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Thesymbols A¹ and A² are independently ═N— or ═CR³—. The symbol L isindependently a bond, —O—, —NH—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, substitutedor unsubstituted heteroarylene,

or —O—(CH₂)_(p)—O—. The symbols k and m are independently 1 or 2. Thesymbol n is independently an integer from 0 to 4. The symbol p isindependently an integer from 1 to 20. The symbol v is independently aninteger from 1 to 20. The symbol w is independently an integer from 1 to20. The symbols z1 and z2 are independently an integer from 0 to 3. Thesymbols X^(1A), X^(1B), X^(2A), X^(2B), and X³ are independently —Cl,—Br, —I, or —F.

In some embodiments, the compound has the formula:

wherein the variables (e.g. R^(1A), R^(1B), R^(2A), R^(2B), R³, A¹, A²,L) are as defined above.

In some embodiments, the compound has the formula:

wherein the variables (e.g. R^(1A), R^(1B), R^(2A), R^(2B), R³, A¹, A²,L) are as defined above.

In some embodiments, the compound has the formula:

wherein the variables (e.g. R^(1A), R^(1B), R^(2A), R^(2B), R³, A¹, A²,L) are as defined above.

In some embodiments, the compound has the formula:

wherein the variables (e.g. R^(1A), R^(1B), R^(2A), R^(2B), R³, A¹, A²,L) are as defined above.

In some embodiments, the compound has the formula:

wherein the variables (e.g. R^(1A), R^(1B), R^(2A), R^(2B), R³, A¹, A²,L) are as defined above.

In some embodiments of the compound of Formula (I), (II), (III), (IV),(V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih) or (IIIh), the compoundis not:

In some embodiments of the compound of Formula (I), (II), (III), (IV),(V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih) or (IIIh), the compoundis selected from:

In some embodiments of the compounds, R^(1A) is —C(O)R^(7A) or—C(O)—OR^(7A). In some embodiments, R^(1A) is —C(O)R^(7A). In someembodiments, R^(1A) is —C(O)—OR^(7A). In some embodiments, R^(2A) is—C(O)R^(9A) or —C(O)—OR^(9A). In some embodiments, R^(2A) is—C(O)R^(9A). In some embodiments, R^(2A) is —C(O)—OR^(9A). In someembodiments, R^(1A) is hydrogen. In some embodiments, R^(2A) ishydrogen. In some embodiments, R^(1A) and R^(2A) are joined to form asubstituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl. In some embodiments, R^(1A) and R^(2A) arejoined to form a substituted or unsubstituted heterocycloalkyl. In someembodiments, R^(1A) and R^(2A) are joined to form a heterocycloalkylfused to an aryl. In some embodiments, R^(1A) and R^(2A) are joined toform a substituted or unsubstituted isoindolin-2-yl-1,3-dione.

In some embodiments of the compounds, R^(1B) is —C(O)R^(7B) or—C(O)—OR^(7B). In some embodiments, R^(1B) is —C(O)R^(7B). In someembodiments, R^(1B) is —C(O)—OR^(7B). In some embodiments, R^(2B) is—C(O)R^(9B) or —C(O)—OR^(9B). In some embodiments, R^(2B) is—C(O)R^(9B). In some embodiments, R^(2B) is —C(O)—OR^(9B). In someembodiments, R^(1B) is hydrogen. In some embodiments, R^(2B) ishydrogen. In some embodiments, R^(1B) and R^(2B) are joined to form asubstituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl. In some embodiments, R^(1B) and R^(2B) arejoined to form a substituted or unsubstituted heterocycloalkyl. In someembodiments, R^(1B) and R^(2B) are joined to form a heterocycloalkylfused to an aryl. In some embodiments, R^(1B) and R^(2B) are joined toform a substituted or unsubstituted isoindolin-2-yl-1,3-dione.

In some embodiments, R^(1A) is substituted or unsubstituted C₁-C₂₀alkyl. In some embodiments, R^(1A) is substituted C₁-C₂₀ alkyl. In someembodiments, R^(1A) is unsubstituted C₁-C₂₀ alkyl. In some embodiments,R^(1A) is substituted or unsubstituted C₁-C₁₂ alkyl. In someembodiments, R^(1A) is substituted C₁-C₁₂ alkyl. In some embodiments,R^(1A) is unsubstituted C₁-C₁₂ alkyl. In some embodiments, R^(1A) issubstituted or unsubstituted C₁-C₅ alkyl. In some embodiments, R^(1A) issubstituted C₁-C₅ alkyl. In some embodiments, R^(1A) is unsubstitutedC₁-C₅ alkyl. In some embodiments, R^(1A) is unsubstituted methyl. Insome embodiments, R^(1A) is unsubstituted ethyl. In some embodiments,R^(1A) is unsubstituted propyl. In some embodiments, R^(1A) istert-butyloxycarbonyl.

In some embodiments, R^(2A) is substituted or unsubstituted C₁-C₂₀alkyl. In some embodiments, R^(2A) is substituted C₁-C₂₀ alkyl. In someembodiments, R^(2A) is unsubstituted C₁-C₂₀ alkyl. In some embodiments,R^(2A) is substituted or unsubstituted C₁-C₁₂ alkyl. In someembodiments, R^(2A) is substituted C₁-C₁₂ alkyl. In some embodiments,R^(2A) is unsubstituted C₁-C₁₂ alkyl. In some embodiments, R^(2A) issubstituted or unsubstituted C₁-C₅ alkyl. In some embodiments, R^(2A) issubstituted C₁-C₅ alkyl. In some embodiments, R^(2A) is unsubstitutedC₁-C₅ alkyl. In some embodiments, R^(2A) is unsubstituted methyl. Insome embodiments, R^(2A) is unsubstituted ethyl. In some embodiments,R^(2A) is unsubstituted propyl. In some embodiments, R^(2A) istert-butyloxycarbonyl.

In some embodiments, R^(1B) is substituted or unsubstituted C₁-C₂₀alkyl. In some embodiments, R^(1B) is substituted C₁-C₂₀ alkyl. In someembodiments, R^(1B) is unsubstituted C₁-C₂₀ alkyl. In some embodiments,R^(1B) is substituted or unsubstituted C₁-C₁₂ alkyl. In someembodiments, R^(1B) is substituted C₁-C₁₂ alkyl. In some embodiments,R^(1B) is unsubstituted C₁-C₁₂ alkyl. In some embodiments, R^(1B) issubstituted or unsubstituted C₁-C₅ alkyl. In some embodiments, R^(1B) issubstituted C₁-C₅ alkyl. In some embodiments, R^(1B) is unsubstitutedC₁-C₅ alkyl. In some embodiments, R^(1B) is unsubstituted methyl. Insome embodiments, R^(1B) is unsubstituted ethyl. In some embodiments,R^(1B) is unsubstituted propyl. In some embodiments, R^(1B) istert-butyloxycarbonyl.

In some embodiments, R^(2B) is substituted or unsubstituted C₁-C₂₀alkyl. In some embodiments, R^(2B) is substituted C₁-C₂₀ alkyl. In someembodiments, R^(2B) is unsubstituted C₁-C₂₀ alkyl. In some embodiments,R^(2B) is substituted or unsubstituted C₁-C₁₂ alkyl. In someembodiments, R^(2B) is substituted C₁-C₁₂ alkyl. In some embodiments,R^(2B) is unsubstituted C₁-C₁₂ alkyl. In some embodiments, R^(2B) issubstituted or unsubstituted C₁-C₅ alkyl. In some embodiments, R^(2B) issubstituted C₁-C₅ alkyl. In some embodiments, R^(2B) is unsubstitutedC₁-C₅ alkyl. In some embodiments, R^(2B) is unsubstituted methyl. Insome embodiments, R^(2B) is unsubstituted ethyl. In some embodiments,R^(2B) is unsubstituted propyl. In some embodiments, R^(2B) istert-butyloxycarbonyl.

In some embodiments of the compounds, R³ is independently hydrogen,—C(O)R¹³, —O—C(O)—R¹³, —C(O)—OR¹³, —OR¹⁴, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In some embodiments, R³ is independently hydrogen, —C(O)R¹³,—O—C(O)—R¹³, —C(O)—OR¹³, or —OR¹⁴. In some embodiments, R³ isindependently halogen. In some embodiments, R³ is independentlyhydrogen. In some embodiments, R³ is independently —C(O)R¹³. In someembodiments, R³ is independently —C(O)—OR¹³. In some embodiments, R³ isindependently —O—C(O)—R¹³. In some embodiments, R³ is independently—OR¹⁴. In some embodiments, R³ is independently —F. In some embodiments,R³ is independently —Cl. In some embodiments, R³ is independently —Br.In some embodiments, R³ is independently —I.

In some embodiments of the compounds, R^(7A) is hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(7A)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(7A)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(7A) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(7A) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(7A) isunsubstituted methyl. In some embodiments, R^(7A) is hydrogen.

In some embodiments of the compounds, R^(8A) is hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(8A)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(8A)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(8A) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(8A) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(8A) isunsubstituted methyl. In some embodiments, R^(8A) is hydrogen.

In some embodiments of the compounds, R^(9A) is hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(9A)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(9A)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(9A) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(9A) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(9A) isunsubstituted methyl. In some embodiments, R^(9A) is hydrogen.

In some embodiments of the compounds, R^(10A) is hydrogen, substitutedor unsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(10A)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(10A)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(10A) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(10A) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(10A) isunsubstituted methyl. In some embodiments, R^(10A) is hydrogen.

In some embodiments of the compounds, R^(7B) is hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(7B)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(7B)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(7B) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(7B) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(7B) isunsubstituted methyl. In some embodiments, R^(7B) is hydrogen.

In some embodiments of the compounds, R^(8B) is hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(8B)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(8B)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(8B) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(8B) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(8B) isunsubstituted methyl. In some embodiments, R^(8B) is hydrogen.

In some embodiments of the compounds, R^(9B) is hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(9B)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(9B)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(9B) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(9B) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(9B) isunsubstituted methyl. In some embodiments, R^(9B) is hydrogen.

In some embodiments of the compounds, R^(10B) is hydrogen, substitutedor unsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 3 to 8 membered heterocycloalkyl,substituted or unsubstituted C₆-C₁₀ aryl, or substituted orunsubstituted 5 to 10 membered heteroaryl. In some embodiments, R^(10B)is hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl or substituted orunsubstituted 2 to 10 membered heteroalkyl. In some embodiments, R^(10B)is hydrogen or substituted or unsubstituted 2 to 10 memberedheteroalkyl. In some embodiments, R^(10B) is hydrogen or substituted orunsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(10B) is substitutedor unsubstituted C₁-C₁₀ alkyl. In some embodiments, R^(10B) isunsubstituted methyl. In some embodiments, R^(10B) is hydrogen.

In some embodiments of the compounds, R¹³ is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In some embodiments, R¹³ isindependently hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl. In some embodiments, R¹³ isindependently hydrogen. In some embodiments, R¹³ is independentlysubstituted or unsubstituted alkyl. In some embodiments, R¹³ isindependently substituted or unsubstituted C₁-C₂₀ alkyl. In someembodiments, R¹³ is independently substituted or unsubstituted C₆-C₁₆alkyl. In some embodiments, R¹³ is independently unsubstituted C₆-C₁₆alkyl. In some embodiments, R¹³ is independently substituted C₆-C₁₆alkyl. In some embodiments, R¹³ is independently substituted orunsubstituted heteroalkyl. In some embodiments, R¹³ is independentlysubstituted or unsubstituted 2 to 20 membered heteroalkyl. In someembodiments, R¹³ is independently substituted or unsubstituted 6 to 16membered heteroalkyl. In some embodiments, R¹³ is independentlysubstituted 6 to 16 membered heteroalkyl. In some embodiments, R¹³ isindependently unsubstituted 6 to 16 membered heteroalkyl. In someembodiments, R¹³ is:

In some embodiments, R¹³ is:

and R³ is independently —C(O)R¹³ or —O—C(O)—R¹³. In some embodiments,R¹³ is independently substituted or unsubstituted C₁₂-C₁₆ alkyl. In someembodiments, R¹³ is independently unsubstituted C₁₂-C₁₆ alkyl. In someembodiments, R¹³ is independently substituted C₁₂-C₁₆ alkyl. In someembodiments, R¹³ is independently substituted or unsubstituted C₁₃-C₁₆alkyl. In some embodiments, R¹³ is independently unsubstituted C₁₃-C₁₆alkyl. In some embodiments, R¹³ is independently substituted C₁₃-C₁₆alkyl. In some embodiments, R¹³ is independently substituted orunsubstituted C₁₄-C₁₆ alkyl. In some embodiments, R¹³ is independentlyunsubstituted C₁₄-C₁₆ alkyl. In some embodiments, R¹³ is independentlysubstituted C₁₄-C₁₆ alkyl. In some embodiments, R¹³ is independentlysubstituted or unsubstituted C₁₅-C₁₆ alkyl. In some embodiments, R¹³ isindependently unsubstituted C₁₅-C₁₆ alkyl. In some embodiments, R¹³ isindependently substituted C₁₅-C₁₆ alkyl. In some embodiments, R¹³ isindependently substituted or unsubstituted C₈-C₁₄ alkyl. In someembodiments, R¹³ is independently unsubstituted C₈-C₁₄ alkyl. In someembodiments, R¹³ is independently substituted C₈-C₁₄ alkyl. In someembodiments, R¹³ is independently substituted or unsubstituted C₆-C₉alkyl. In some embodiments, R¹³ is independently unsubstituted C₆-C₉alkyl. In some embodiments, R¹³ is independently substituted C₆-C₉alkyl. In some embodiments, R¹³ is independently substituted orunsubstituted C₉ alkyl. In some embodiments, R¹³ is independentlyunsubstituted C₉ alkyl. In some embodiments, R¹³ is independentlysubstituted C₉ alkyl. In some embodiments, R¹³ is independentlysubstituted or unsubstituted C₃-C₂₀ branched alkyl. In some embodiments,R¹³ is independently unsubstituted C₃-C₂₀ branched alkyl. In someembodiments, R¹³ is independently substituted C₃-C₂₀ branched alkyl. Insome embodiments, R¹³ is independently substituted or unsubstitutedC₆-C₁₆ branched alkyl. In some embodiments, R¹³ is independentlyunsubstituted C₆-C₁₆ branched alkyl. In some embodiments, R¹³ isindependently substituted C₆-C₁₆ branched alkyl. In some embodiments,R¹³ is independently substituted or unsubstituted C₆-C₁₂ branched alkyl.In some embodiments, R¹³ is independently unsubstituted C₆-C₁₂ branchedalkyl. In some embodiments, R¹³ is independently substituted C₆-C₁₂branched alkyl. In some embodiments, R¹³ is independently substituted orunsubstituted C₉-C₁₆ branched alkyl. In some embodiments, R¹³ isindependently unsubstituted C₉-C₁₆ branched alkyl. In some embodiments,R¹³ is independently substituted C₉-C₁₆ branched alkyl. In someembodiments, R¹³ is independently substituted or unsubstituted C₉-C₁₂branched alkyl. In some embodiments, R¹³ is independently unsubstitutedC₉-C₁₂ branched alkyl. In some embodiments, R¹³ is independentlysubstituted C₉-C₁₂ branched alkyl. In some embodiments, R¹³ isindependently substituted or unsubstituted 12 to 20 memberedheteroalkyl. In some embodiments, R¹³ is independently substituted 12 to20 membered heteroalkyl. In some embodiments, R¹³ is independentlyunsubstituted 12 to 20 membered heteroalkyl. In some embodiments, R¹³ isindependently substituted or unsubstituted 13 to 20 memberedheteroalkyl. In some embodiments, R¹³ is independently substituted 13 to20 membered heteroalkyl. In some embodiments, R¹³ is independentlyunsubstituted 13 to 20 membered heteroalkyl. In some embodiments, R¹³ isindependently substituted or unsubstituted 14 to 20 memberedheteroalkyl. In some embodiments, R¹³ is independently substituted 14 to20 membered heteroalkyl. In some embodiments, R¹³ is independentlyunsubstituted 14 to 20 membered heteroalkyl. In some embodiments, R¹³ isindependently substituted or unsubstituted 15 to 20 memberedheteroalkyl. In some embodiments, R¹³ is independently substituted 15 to20 membered heteroalkyl. In some embodiments, R¹³ is independentlyunsubstituted 15 to 20 membered heteroalkyl. In some embodiments, R¹³ issubstituted or unsubstituted C₃-C₈ cycloalkyl. In some embodiments, R¹³is substituted or unsubstituted 3 to 8 membered heterocycloalkyl. Insome embodiments, R¹³ is substituted or unsubstituted C₆-C₁₀ aryl. Insome embodiments, R¹³ is substituted or unsubstituted 5 to 10 memberedheteroaryl. In some embodiments, R¹³ is substituted or unsubstituted 5to 6 membered heteroaryl. In some embodiments, R¹³ is unsubstitutedethyl. In some embodiments, R¹³ is unsubstituted propyl. In someembodiments, R¹³ is unsubstituted methyl.

In some embodiments of the compounds, R¹⁴ is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In some embodiments, R¹⁴ isindependently hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl. In some embodiments, R¹⁴ isindependently hydrogen. In some embodiments, R¹⁴ is independentlysubstituted or unsubstituted alkyl. In some embodiments, R¹⁴ isindependently substituted or unsubstituted C₁-C₂₀ alkyl. In someembodiments, R¹⁴ is independently substituted or unsubstituted C₆-C₁₆alkyl. In some embodiments, R¹⁴ is independently unsubstituted C₆-C₁₆alkyl. In some embodiments, R¹⁴ is independently substituted C₆-C₁₆alkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted heteroalkyl. In some embodiments, R¹⁴ is independentlysubstituted or unsubstituted 2 to 20 membered heteroalkyl. In someembodiments, R¹⁴ is independently substituted or unsubstituted 6 to 16membered heteroalkyl. In some embodiments, R¹⁴ is independentlysubstituted 6 to 16 membered heteroalkyl. In some embodiments, R¹⁴ isindependently unsubstituted 6 to 16 membered heteroalkyl. In someembodiments, R¹⁴ is:

In some embodiments, R¹⁴ is independently substituted or unsubstitutedC₁₂-C₁₆ alkyl. In some embodiments, R¹⁴ is independently unsubstitutedC₁₂-C₁₆ alkyl. In some embodiments, R¹⁴ is independently substitutedC₁₂-C₁₆ alkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted C₁₃-C₁₆ alkyl. In some embodiments, R¹⁴ is independentlyunsubstituted C₁₃-C₁₆ alkyl. In some embodiments, R¹⁴ is independentlysubstituted C₁₃-C₁₆ alkyl. In some embodiments, R¹⁴ is independentlysubstituted or unsubstituted C₁₄-C₁₆ alkyl. In some embodiments, R¹⁴ isindependently unsubstituted C₁₄-C₁₆ alkyl. In some embodiments, R¹⁴ isindependently substituted C₁₄-C₁₆ alkyl. In some embodiments, R¹⁴ isindependently substituted or unsubstituted C₁₅-C₁₆ alkyl. In someembodiments, R¹⁴ is independently unsubstituted C₁₅-C₁₆ alkyl. In someembodiments, R¹⁴ is independently substituted C₁₅-C₁₆ alkyl. In someembodiments, R¹⁴ is independently substituted or unsubstituted C₈-C₁₄alkyl. In some embodiments, R¹⁴ is independently unsubstituted C₈-C₁₄alkyl. In some embodiments, R¹⁴ is independently substituted C₈-C₁₄alkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted C₆-C₉ alkyl. In some embodiments, R¹⁴ is independentlyunsubstituted C₆-C₉ alkyl. In some embodiments, R¹⁴ is independentlysubstituted C₆-C₉ alkyl. In some embodiments, R¹⁴ is independentlysubstituted or unsubstituted C₉ alkyl. In some embodiments, R¹⁴ isindependently unsubstituted C₉ alkyl. In some embodiments, R¹⁴ isindependently substituted C₉ alkyl. In some embodiments, R¹⁴ isindependently substituted or unsubstituted C₃-C₂₀ branched alkyl. Insome embodiments, R¹⁴ is independently unsubstituted C₃-C₂₀ branchedalkyl. In some embodiments, R¹⁴ is independently substituted C₃-C₂₀branched alkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted C₆-C₁₆ branched alkyl. In some embodiments, R¹⁴ isindependently unsubstituted C₆-C₁₆ branched alkyl. In some embodiments,R¹⁴ is independently substituted C₆-C₁₆ branched alkyl. In someembodiments, R¹⁴ is independently substituted or unsubstituted C₆-C₁₂branched alkyl. In some embodiments, R¹⁴ is independently unsubstitutedC₆-C₁₂ branched alkyl. In some embodiments, R¹⁴ is independentlysubstituted C₆-C₁₂ branched alkyl. In some embodiments, R¹⁴ isindependently substituted or unsubstituted C₉-C₁₆ branched alkyl. Insome embodiments, R¹⁴ is independently unsubstituted C₉-C₁₆ branchedalkyl. In some embodiments, R¹⁴ is independently substituted C₉-C₁₆branched alkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted C₉-C₁₂ branched alkyl. In some embodiments, R¹⁴ isindependently unsubstituted C₉-C₁₂ branched alkyl. In some embodiments,R¹⁴ is independently substituted C₉-C₁₂ branched alkyl. In someembodiments, R¹⁴ is independently substituted or unsubstituted C₈-C₁₂alkyl. In some embodiments, R¹⁴ is independently substituted C₈-C₁₂alkyl. In some embodiments, R¹⁴ is independently unsubstituted C₈-C₁₂alkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted 12 to 20 membered heteroalkyl. In some embodiments, R¹⁴ isindependently substituted 12 to 20 membered heteroalkyl. In someembodiments, R¹⁴ is independently unsubstituted 12 to 20 memberedheteroalkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted 13 to 20 membered heteroalkyl. In some embodiments, R¹⁴ isindependently substituted 13 to 20 membered heteroalkyl. In someembodiments, R¹⁴ is independently unsubstituted 13 to 20 memberedheteroalkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted 14 to 20 membered heteroalkyl. In some embodiments, R¹⁴ isindependently substituted 14 to 20 membered heteroalkyl. In someembodiments, R¹⁴ is independently unsubstituted 14 to 20 memberedheteroalkyl. In some embodiments, R¹⁴ is independently substituted orunsubstituted 15 to 20 membered heteroalkyl. In some embodiments, R¹⁴ isindependently substituted 15 to 20 membered heteroalkyl. In someembodiments, R¹⁴ is independently unsubstituted 15 to 20 memberedheteroalkyl. In some embodiments, R¹⁴ is substituted or unsubstitutedC₃-C₈ cycloalkyl. In some embodiments, R¹⁴ is unsubstituted C₃-C₈cycloalkyl. In some embodiments, R¹⁴ is substituted C₃-C₈ cycloalkyl. Insome embodiments, R¹⁴ is substituted or unsubstituted 3 to 8 memberedheterocycloalkyl. In some embodiments, R¹⁴ is unsubstituted 3 to 8membered heterocycloalkyl. In some embodiments, R¹⁴ is substituted 3 to8 membered heterocycloalkyl. In some embodiments, R¹⁴ is substituted orunsubstituted C₆-C₁₀ aryl. In some embodiments, R¹⁴ is unsubstitutedC₆-C₁₀ aryl. In some embodiments, R¹⁴ is substituted C₆-C₁₀ aryl. Insome embodiments, R¹⁴ is substituted or unsubstituted 5 to 10 memberedheteroaryl. In some embodiments, R¹⁴ is unsubstituted 5 to 10 memberedheteroaryl. In some embodiments, R¹⁴ is substituted 5 to 10 memberedheteroaryl. In some embodiments, R¹⁴ is substituted or unsubstituted 5to 6 membered heteroaryl. In some embodiments, R¹⁴ is unsubstituted 5 to6 membered heteroaryl. In some embodiments, R¹⁴ is substituted 5 to 6membered heteroaryl. In some embodiments, R¹⁴ is unsubstituted ethyl. Insome embodiments, R¹⁴ is unsubstituted propyl. In some embodiments, R¹⁴is unsubstituted methyl.

R⁴ is independently hydrogen, halogen, —CX⁴ ₃, —CN, —SO₂Cl, —SO_(q)R¹⁸,—SO_(r)NR¹⁵R¹⁶, —NHNH₂, —ONR¹⁵R¹⁶, —NHC═(O)NHNH₂, —NHC═(O)NR¹⁵R¹⁶,—N(O)_(t), —NR¹⁵R¹⁶, —C(O)R¹⁷, —C(O)—OR¹⁷, —C(O)NR¹⁵R¹⁶, —OR¹⁸,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted membered heteroaryl. R¹⁵, R¹⁶, R¹⁷, and R¹⁸are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Thesymbols r and t are independently 1 or 2. The symbol q is independentlyan integer from 0 to 4. The symbol u is independently an integer from 0to 5. The symbol X⁴ is independently —Cl, —Br, —I, or —F.

In some embodiments of the compounds, the symbol L is

In some embodiments of the compounds, the symbol L

In some embodiments of the compounds, the symbol L is

In some embodiments of the compounds, the symbol L is

In some embodiments of the compounds, the symbol L is

In some embodiments of the compounds, the symbol L is —O—(CH₂)_(p)—O—.In some embodiments of the compounds, the symbol L is

In some embodiments of the compounds, the symbol L is

In some embodiments of the compounds, the symbol L is a bond. In someembodiments of the compounds, the symbol L is —O—. In some embodimentsof the compounds, the symbol L is —NH—. In some embodiments of thecompounds, the symbol L is substituted or unsubstituted alkylene. Insome embodiments of the compounds, the symbol L is substituted orunsubstituted heteroalkylene. In some embodiments of the compounds, thesymbol L is substituted or unsubstituted cycloalkylene. In someembodiments of the compounds, the symbol L is substituted orunsubstituted heterocycloalkylene. In some embodiments of the compounds,the symbol L is substituted or unsubstituted arylene. In someembodiments of the compounds, the symbol L is substituted orunsubstituted heteroarylene. In some embodiments of the compounds, thesymbol L is unsubstituted alkylene. In some embodiments of thecompounds, the symbol L is unsubstituted heteroalkylene. In someembodiments of the compounds, the symbol L is unsubstitutedcycloalkylene. In some embodiments of the compounds, the symbol L isunsubstituted heterocycloalkylene. In some embodiments of the compounds,the symbol L is unsubstituted arylene. In some embodiments of thecompounds, the symbol L is unsubstituted heteroarylene. In someembodiments of the compounds, the symbol L is substituted orunsubstituted C₁-C₂₀ alkylene. In some embodiments of the compounds, thesymbol L is substituted or unsubstituted C₈-C₁₆ alkylene. In someembodiments of the compounds, the symbol L is substituted orunsubstituted C₁₀-C₁₄ alkylene. In some embodiments of the compounds,the symbol L is substituted C₁₀-C₁₄ alkylene. In some embodiments of thecompounds, the symbol L is unsubstituted C₁₀-C₁₄ alkylene. In someembodiments of the compounds, the symbol L is unsubstituted C₁₂alkylene. In some embodiments of the compounds, the symbol L is asubstituted or unsubstituted 2 to 20 membered heteroalkylene. In someembodiments of the compounds, the symbol L is a substituted orunsubstituted C₃-C₈ cycloalkylene. In some embodiments of the compounds,the symbol L is a substituted or unsubstituted 3 to 8 memberedheterocycloalkylene. In some embodiments of the compounds, the symbol Lis substituted or unsubstituted C₆-C₁₀ arylene. In some embodiments ofthe compounds, the symbol L is a substituted or unsubstituted 5 to 10membered heteroarylene. In some embodiments of the compounds, the symbolL is substituted or unsubstituted C₁-C₈ alkylene. In some embodiments ofthe compounds, the symbol L is a substituted or unsubstituted 2 to 8membered heteroalkylene. In some embodiments of the compounds, thesymbol L is a substituted or unsubstituted C₃-C₇ cycloalkylene. In someembodiments of the compounds, the symbol L is a substituted orunsubstituted 3 to 7 membered heterocycloalkylene. In some embodimentsof the compounds, the symbol L is a substituted or unsubstituted 5 to 9membered heteroarylene.

In some embodiments of the compounds, the symbol A¹ is ═CH—. In someembodiments of the compounds, the symbol A¹ is ═N—. In some embodimentsof the compounds, the symbol A² is ═CH—. In some embodiments of thecompounds, the symbol A² is ═N—. In some embodiments of the compounds,the symbol A² is ═CR³—. In some embodiments of the compounds, the symbolA¹ is ═CR³—.

In some embodiments, k is independently 1. In some embodiments, k isindependently 2. In some embodiments, m is independently 1. In someembodiments, m is independently 2. In some embodiments, n isindependently 0. In some embodiments, n is independently 1. In someembodiments, n is independently 2. In some embodiments, n isindependently 3. In some embodiments, n is independently 4. In someembodiments, the symbol X^(1A) is independently —Cl. In someembodiments, the symbol X^(1A) is independently —Br. In someembodiments, the symbol X^(1A) is independently —I. In some embodiments,the symbol X^(1A) is independently —F. In some embodiments, the symbolX^(2A) is independently —Cl. In some embodiments, the symbol X^(2A) isindependently —Br. In some embodiments, the symbol X^(2A) isindependently —I. In some embodiments, the symbol X^(2A) isindependently —F. In some embodiments, the symbol X^(1B) isindependently —Cl. In some embodiments, the symbol X^(1B) isindependently —Br. In some embodiments, the symbol X^(1B) isindependently —I. In some embodiments, the symbol X^(1B) isindependently —F. In some embodiments, the symbol X^(2B) isindependently —Cl. In some embodiments, the symbol X^(2B) isindependently —Br. In some embodiments, the symbol X^(2B) isindependently —I. In some embodiments, the symbol X^(2B) isindependently —F. In some embodiments, the symbol X³ is independently—Cl. In some embodiments, the symbol X³ is independently —Br. In someembodiments, the symbol X³ is independently —I. In some embodiments, thesymbol X³ is independently —F. In some embodiments, r isindependently 1. In some embodiments, r is independently 2. In someembodiments, t is independently 1. In some embodiments, t isindependently 2. In some embodiments, q is independently 0. In someembodiments, q is independently 1. In some embodiments, q isindependently 2. In some embodiments, q is independently 3. In someembodiments, q is independently 4. In some embodiments, the symbol X⁴ isindependently —Cl. In some embodiments, the symbol X⁴ is independently—Br. In some embodiments, the symbol X⁴ is independently —I. In someembodiments, the symbol X⁴ is independently —F. In some embodiments, uis independently 0. In some embodiments, u is independently 1. In someembodiments, u is independently 2. In some embodiments, u isindependently 3. In some embodiments, u is independently 4. In someembodiments, u is independently 5. In some embodiments, the symbol p isindependently 1. In some embodiments, the symbol p is independently 2.In some embodiments, the symbol p is independently 3. In someembodiments, the symbol p is independently 4. In some embodiments, thesymbol p is independently 5. In some embodiments, the symbol p isindependently 6. In some embodiments, the symbol p is independently 7.In some embodiments, the symbol p is independently 8. In someembodiments, the symbol p is independently 9. In some embodiments, thesymbol p is independently 10. In some embodiments, the symbol p isindependently 11. In some embodiments, the symbol p is independently 12.In some embodiments, the symbol p is independently 13. In someembodiments, the symbol p is independently 14. In some embodiments, thesymbol p is independently 15. In some embodiments, the symbol p isindependently 16. In some embodiments, the symbol p is independently 17.In some embodiments, the symbol p is independently 18. In someembodiments, the symbol p is independently 19. In some embodiments, thesymbol p is independently 20. In some embodiments, the symbol v isindependently 1. In some embodiments, the symbol v is independently 2.In some embodiments, the symbol v is independently 3. In someembodiments, the symbol v is independently 4. In some embodiments, thesymbol v is independently 5. In some embodiments, the symbol v isindependently 6. In some embodiments, the symbol v is independently 7.In some embodiments, the symbol v is independently 8. In someembodiments, the symbol v is independently 9. In some embodiments, thesymbol v is independently 10. In some embodiments, the symbol v isindependently 11. In some embodiments, the symbol v is independently 12.In some embodiments, the symbol v is independently 13. In someembodiments, the symbol v is independently 14. In some embodiments, thesymbol v is independently 15. In some embodiments, the symbol v isindependently 16. In some embodiments, the symbol v is independently 17.In some embodiments, the symbol v is independently 18. In someembodiments, the symbol v is independently 19. In some embodiments, thesymbol v is independently 20. In some embodiments, the symbol w isindependently 1. In some embodiments, the symbol w is independently 2.In some embodiments, the symbol w is independently 3. In someembodiments, the symbol w is independently 4. In some embodiments, thesymbol w is independently 5. In some embodiments, the symbol w isindependently 6. In some embodiments, the symbol w is independently 7.In some embodiments, the symbol w is independently 8. In someembodiments, the symbol w is independently 9. In some embodiments, thesymbol w is independently 10. In some embodiments, the symbol w isindependently 11. In some embodiments, the symbol w is independently 12.In some embodiments, the symbol w is independently 13. In someembodiments, the symbol w is independently 14. In some embodiments, thesymbol w is independently 15. In some embodiments, the symbol w isindependently 16. In some embodiments, the symbol w is independently 17.In some embodiments, the symbol w is independently 18. In someembodiments, the symbol w is independently 19. In some embodiments, thesymbol w is independently 20. In some embodiments, z1 is independently0. In some embodiments, z1 is independently 1. In some embodiments, z1is independently 2. In some embodiments, z1 is independently 3. In someembodiments, z2 is independently 0. In some embodiments, z2 isindependently 1. In some embodiments, z2 is independently 2. In someembodiments, z2 is independently 3.

In some embodiments of the compounds, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₂-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₃-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₄-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₅-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₆-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is halogensubstituted C₁-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is halogensubstituted C₃-C₁₆ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is halogensubstituted C₆-C₁₂ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is halogensubstituted C₆-C₉ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —CNsubstituted C₁-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —CNsubstituted C₃-C₁₆ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —CNsubstituted C₆-C₁₂ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —CNsubstituted C₆-C₉ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ isunsubstituted branched C₁-C₂₀ alkyl. In some embodiments, the symbol A¹is ═CH—, R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ isunsubstituted branched C₃-C₁₆ alkyl. In some embodiments, the symbol A¹is ═CH—, R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ isunsubstituted branched C₆-C₁₂ alkyl. In some embodiments, the symbol A¹is ═CH—, R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ isunsubstituted branched C₆-C₉ alkyl. In some embodiments, the symbol A¹is ═CH—, R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ isunsubstituted branched C₇-C₈ alkyl. In some embodiments, the symbol A¹is ═CH—, R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is—(CH₂CH₂O)₁₋₁₀CH₃. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —(CH₂CH₂O)₁₋₈CH₃.In some embodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A)is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —(CH₂CH₂O)₁₋₆CH₃. In someembodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) ishydrogen, R³ is —OR¹⁴, and R¹⁴ is —(CH₂CH₂O)₁₋₄CH₃. In some embodiments,the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is—OR¹⁴, and R¹⁴ is —(CH₂CH₂O)₁₋₃CH₃. In some embodiments, the symbol A¹is ═CH—, R^(2A) is hydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is—(CH₂CH₂O)₃₋₁₀CH₃. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —(CH₂CH₂O)₃₋₈CH₃.In some embodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A)is hydrogen, R³ is —OR¹⁴, and R¹⁴ is —(CH₂CH₂O)₃₋₆CH₃.

In some embodiments of the compounds, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₂-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₃-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₄-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₅-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₆-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₂-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₂-C₁₆ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₂-C₁₂ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₂-C₉ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₃-C₁₂ alkyl. In some embodiments, the symbol A¹ is ═N—, R^(2A) ishydrogen, R^(1A) is hydrogen, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₆-C₁₂ alkyl.

In some embodiments of the compounds, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₂-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₃-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₄-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₅-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₆-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is substitutedC₁₂-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is substitutedC₁₃-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is substitutedC₁₄-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is substitutedC₁₅-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)CH₃, R³ is —OR¹⁴, and R¹⁴ is substitutedC₁₆-C₂₀ alkyl.

In some embodiments of the compounds, the symbol A¹ is ═CH—, R^(2A) isunsubstituted methyl, R^(1A) is unsubstituted methyl, R³ is —OR¹⁴, andR¹⁴ is hydrogen. In some embodiments, the symbol A¹ is ═CH—, R^(2A) isunsubstituted methyl, R^(1A) is unsubstituted methyl, R³ is —OR¹⁴, andR¹⁴ is unsubstituted C₂-C₂₀ alkyl. In some embodiments, the symbol A¹ is═CH—, R^(2A) is unsubstituted methyl, R^(1A) is unsubstituted methyl, R³is —OR¹⁴, and R¹⁴ is unsubstituted C₂-C₁₆ alkyl. In some embodiments,the symbol A¹ is ═CH—, R^(2A) is unsubstituted methyl, R^(1A) isunsubstituted methyl, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₂-C₁₂alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) isunsubstituted methyl, R^(1A) is unsubstituted methyl, R³ is —OR¹⁴, andR¹⁴ is unsubstituted C₂-C₈ alkyl. In some embodiments, the symbol A¹ is═CH—, R^(2A) is unsubstituted methyl, R^(1A) is unsubstituted methyl, R³is —OR¹⁴, and R¹⁴ is unsubstituted C₂-C₆ alkyl. In some embodiments, thesymbol A¹ is ═CH—, R^(2A) is unsubstituted methyl, R^(1A) isunsubstituted methyl, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₄-C₂₀alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) isunsubstituted methyl, R^(1A) is unsubstituted methyl, R³ is —OR¹⁴, andR¹⁴ is unsubstituted C₆-C₂₀ alkyl. In some embodiments, the symbol A¹ is═CH—, R^(2A) is unsubstituted methyl, R^(1A) is unsubstituted methyl, R³is —OR¹⁴, and R¹⁴ is unsubstituted C₈-C₂₀ alkyl. In some embodiments,the symbol A¹ is ═CH—, R^(2A) is unsubstituted methyl, R^(1A) isunsubstituted methyl, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₁₀-C₂₀alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) isunsubstituted methyl, R^(1A) is unsubstituted methyl, R³ is —OR¹⁴, andR¹⁴ is unsubstituted C₁₂-C₂₀ alkyl. In some embodiments, the symbol A¹is ═CH—, R^(2A) is unsubstituted methyl, R^(1A) is unsubstituted methyl,R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₁₄-C₂₀ alkyl. In someembodiments, the symbol A¹ is ═CH—, R^(2A) is unsubstituted methyl,R^(1A) is unsubstituted methyl, R³ is —OR¹⁴, and R¹⁴ is unsubstitutedC₁₆-C₂₀ alkyl.

In some embodiments of the compounds, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is —C(O)R^(7A), R^(7A) is unsubstituted C₈-C₁₀ alkyl,R³ is —OC(O)R¹³, and R¹³ is unsubstituted C₁-C₂₀ alkyl. In someembodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is—C(O)R^(7A), R^(7A) is unsubstituted C₈-C₁₀ alkyl, R³ is —OC(O)R¹³, andR¹³ is unsubstituted C₁-C₁₆ alkyl. In some embodiments, the symbol A¹ is═CH—, R^(2A) is hydrogen, R^(1A) is —C(O)R^(7A), R^(7A) is unsubstitutedC₈-C₁₀ alkyl, R³ is —OC(O)R¹³, and R¹³ is unsubstituted C₁-C₁₂ alkyl. Insome embodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is—C(O)R^(7A), R^(7A) is unsubstituted C₈-C₁₀ alkyl, R³ is —OC(O)R¹³, andR¹³ is unsubstituted C₁-C₉ alkyl. In some embodiments, the symbol A¹ is═CH—, R^(2A) is hydrogen, R^(1A) is —C(O)R^(7A), R^(7A) is unsubstitutedC₈-C₁₀ alkyl, R³ is —OC(O)R¹³, and R¹³ is unsubstituted C₄-C₂₀ alkyl. Insome embodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is—C(O)R^(7A), R^(7A) is unsubstituted C₈-C₁₀ alkyl, R³ is —OC(O)R¹³, andR¹³ is unsubstituted C₈-C₂₀ alkyl. In some embodiments, the symbol A¹ is═CH—, R^(2A) is hydrogen, R^(1A) is —C(O)R^(7A), R^(7A) is unsubstitutedC₈-C₁₀ alkyl, R³ is —OC(O)R¹³, and R¹³ is unsubstituted C₈-C₁₆ alkyl. Insome embodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is—C(O)R^(7A), R^(7A) is unsubstituted C₈-C₁₀ alkyl, R³ is —OC(O)R¹³, andR¹³ is unsubstituted C₈-C₁₂ alkyl. In some embodiments, the symbol A¹ is═CH—, R^(2A) is hydrogen, R^(1A) is —C(O)R^(7A), R^(7A) is unsubstitutedC₈-C₁₀ alkyl, and R³ hydrogen.

In some embodiments of the compounds, the symbol A¹ is ═CH—, R^(2A) ishydrogen, R^(1A) is unsubstituted C₉-C₁₂ alkyl, R³ is —OR¹⁴, and R¹⁴ isunsubstituted C₁-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is unsubstituted C₉-C₁₂ alkyl, R³ is —OR¹⁴,and R¹⁴ is unsubstituted C₁-C₁₆ alkyl. In some embodiments, the symbolA¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is unsubstituted C₉-C₁₂ alkyl, R³is —OR¹⁴, and R¹⁴ is unsubstituted C₁-C₁₂ alkyl. In some embodiments,the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is unsubstitutedC₉-C₁₂ alkyl, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₁-C₉ alkyl. In someembodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) isunsubstituted C₉-C₁₂ alkyl, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₁-C₆alkyl. In some embodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen,R^(1A) is unsubstituted C₉-C₁₂ alkyl, R³ is —OR¹⁴, and R¹⁴ isunsubstituted C₄-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═CH—,R^(2A) is hydrogen, R^(1A) is unsubstituted C₉-C₁₂ alkyl, R³ is —OR¹⁴,and R¹⁴ is unsubstituted C₈-C₂₀ alkyl. In some embodiments, the symbolA¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is unsubstituted C₉-C₁₂ alkyl, R³is —OR¹⁴, and R¹⁴ is unsubstituted C₈-C₁₆ alkyl. In some embodiments,the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is unsubstitutedC₉-C₁₂ alkyl, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₈-C₁₂ alkyl. Insome embodiments, the symbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) isunsubstituted C₉-C₁₂ alkyl, and R³ is —OH. In some embodiments, thesymbol A¹ is ═CH—, R^(2A) is hydrogen, R^(1A) is unsubstituted C₉-C₁₂alkyl, and R³ is hydrogen.

In some embodiments of the compounds, the symbol A¹ is ═N—, R^(1A) andR^(2A) are joined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³is —OR¹⁴, and R¹⁴ is unsubstituted C₁-C₂₀ alkyl. In some embodiments,the symbol A¹ is ═N—, R^(1A) and R^(2A) are joined to form anunsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ isunsubstituted C₃-C₂₀ alkyl. In some embodiments, the symbol A¹ is ═N—,R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₆-C₂₀alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is unsubstituted C₉-C₂₀ alkyl. In some embodiments, the symbolA¹ is ═N—, R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₁-C₁₆alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is unsubstituted C₁-C₁₂ alkyl. In some embodiments, the symbolA¹ is ═N—, R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₁-C₉alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is unsubstituted C₆-C₁₂ alkyl. In some embodiments, the symbolA¹ is ═N—, R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is unsubstituted C₈-C₁₀alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is substituted C₁-C₂₀ alkyl. In some embodiments, the symbol A¹is ═N—, R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is substituted C₃-C₂₀alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is substituted C₆-C₂₀ alkyl. In some embodiments, the symbol A¹is ═N—, R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is substituted C₉-C₂₀alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is substituted C₁-C₁₆ alkyl. In some embodiments, the symbol A¹is ═N—, R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is substituted C₁-C₁₂alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is substituted C₁-C₉ alkyl. In some embodiments, the symbol A¹is ═N—, R^(1A) and R^(2A) are joined to form an unsubstitutedisoindolin-2-yl-1,3-dione, R³ is —OR¹⁴, and R¹⁴ is substituted C₆-C₁₂alkyl. In some embodiments, the symbol A¹ is ═N—, R^(1A) and R^(2A) arejoined to form an unsubstituted isoindolin-2-yl-1,3-dione, R³ is —OR¹⁴,and R¹⁴ is substituted C₈-C₁₀ alkyl.

In some embodiments of the compounds provided herein, R^(1A) isindependently hydrogen, halogen, —CF₃, —COOH, —CONH₂, —C(O)CH₃,R^(20A)-substituted or unsubstituted alkyl, R^(20A)-substituted orunsubstituted heteroalkyl, R^(20A)-substituted or unsubstitutedcycloalkyl, R^(20A)-substituted or unsubstituted heterocycloalkyl,R^(20A)-substituted or unsubstituted aryl, or R^(20A)-substituted orunsubstituted heteroaryl.

R^(20A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(21A)-substituted or unsubstituted alkyl, R^(21A)-substituted orunsubstituted heteroalkyl, R^(21A)-substituted or unsubstitutedcycloalkyl, R^(21A)-substituted or unsubstituted heterocycloalkyl,R^(21A)-substituted or unsubstituted aryl, or R^(21A)-substituted orunsubstituted heteroaryl.

R^(21A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(22A)-substituted or unsubstituted alkyl, R^(22A)-substituted orunsubstituted heteroalkyl, R^(22A)-substituted or unsubstitutedcycloalkyl, R^(22A)-substituted or unsubstituted heterocycloalkyl,R^(22A)-substituted or unsubstituted aryl, or R^(22A)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(1B) isindependently hydrogen, halogen, —CF₃, —COOH, —CONH₂, —C(O)CH₃,R^(20B)-substituted or unsubstituted alkyl, R^(20B)-substituted orunsubstituted heteroalkyl, R^(20B)-substituted or unsubstitutedcycloalkyl, R^(20B)-substituted or unsubstituted heterocycloalkyl,R^(20B)-substituted or unsubstituted aryl, or R^(20B)-substituted orunsubstituted heteroaryl.

R^(20B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(21B)-substituted or unsubstituted alkyl, R^(21B)-substituted orunsubstituted heteroalkyl, R^(21B)-substituted or unsubstitutedcycloalkyl, R^(21B)-substituted or unsubstituted heterocycloalkyl,R^(21B)-substituted or unsubstituted aryl, or R^(21B)-substituted orunsubstituted heteroaryl.

R^(21B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(22B)-substituted or unsubstituted alkyl, R^(22B)-substituted orunsubstituted heteroalkyl, R^(22B)-substituted or unsubstitutedcycloalkyl, R^(22B)-substituted or unsubstituted heterocycloalkyl,R^(22B)-substituted or unsubstituted aryl, or R^(22B)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(2A) isindependently hydrogen, halogen, —CF₃, —COOH, —CONH₂, —C(O)CH₃,R^(23A)-substituted or unsubstituted alkyl, R^(23A)-substituted orunsubstituted heteroalkyl, R^(23A)-substituted or unsubstitutedcycloalkyl, R^(23A)-substituted or unsubstituted heterocycloalkyl,R^(23A)-substituted or unsubstituted aryl, or R^(23A)-substituted orunsubstituted heteroaryl.

R^(23A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(24A)-substituted or unsubstituted alkyl, R^(24A)-substituted orunsubstituted heteroalkyl, R^(24A)-substituted or unsubstitutedcycloalkyl, R^(24A)-substituted or unsubstituted heterocycloalkyl,R^(24A)-substituted or unsubstituted aryl, or R^(24A)-substituted orunsubstituted heteroaryl.

R^(24A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(25A)-substituted or unsubstituted alkyl, R^(25A)-substituted orunsubstituted heteroalkyl, R^(25A)-substituted or unsubstitutedcycloalkyl, R^(25A)-substituted or unsubstituted heterocycloalkyl,R^(25A)-substituted or unsubstituted aryl, or R^(25A)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(2B) isindependently hydrogen, halogen, —CF₃, —COOH, —CONH₂, —C(O)CH₃,R^(23B)-substituted or unsubstituted alkyl, R^(23B)-substituted orunsubstituted heteroalkyl, R^(23B)-substituted or unsubstitutedcycloalkyl, R^(23B)-substituted or unsubstituted heterocycloalkyl,R^(23B)-substituted or unsubstituted aryl, or R^(23B)-substituted orunsubstituted heteroaryl.

R^(23B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(24B)-substituted or unsubstituted alkyl, R^(24B)-substituted orunsubstituted heteroalkyl, R^(24B)-substituted or unsubstitutedcycloalkyl, R^(24B)-substituted or unsubstituted heterocycloalkyl,R^(24B)-substituted or unsubstituted aryl, or R^(24B)-substituted orunsubstituted heteroaryl.

R^(24B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(25B)-substituted or unsubstituted alkyl, R^(25B)-substituted orunsubstituted heteroalkyl, R^(25B)-substituted or unsubstitutedcycloalkyl, R^(25B)-substituted or unsubstituted heterocycloalkyl,R^(25B)-substituted or unsubstituted aryl, or R^(25B)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R³ isindependently hydrogen, halogen, —CF₃, —CN, —OH, —NH₂, —COOH,—OC(O)H—CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, R²⁶-substituted or unsubstituted alkyl, R²⁶-substitutedor unsubstituted heteroalkyl, R²⁶-substituted or unsubstitutedcycloalkyl, R²⁶-substituted or unsubstituted heterocycloalkyl,R²⁶-substituted or unsubstituted aryl, or R²⁶-substituted orunsubstituted heteroaryl.

R²⁶ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R²⁷-substituted or unsubstituted alkyl, R²⁷-substituted or unsubstitutedheteroalkyl, R²⁷-substituted or unsubstituted cycloalkyl, R²⁷substitutedor unsubstituted heterocycloalkyl, R²⁷-substituted or unsubstitutedaryl, or R²⁷-substituted or unsubstituted heteroaryl.

R²⁷ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R²⁸-substituted or unsubstituted alkyl, R²⁸-substituted or unsubstitutedheteroalkyl, R²⁸-substituted or unsubstituted cycloalkyl,R²⁸-substituted or unsubstituted heterocycloalkyl, R²⁸-substituted orunsubstituted aryl, or R²⁸-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R⁴ isindependently hydrogen, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,—C(O)OCH₃, R²⁹-substituted or unsubstituted alkyl, R²⁹-substituted orunsubstituted heteroalkyl, R²⁹-substituted or unsubstituted cycloalkyl,R²⁹-substituted or unsubstituted 4 heterocycloalkyl, R²⁹-substituted orunsubstituted aryl, or R²⁹-substituted or unsubstituted heteroaryl.

R²⁹ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R³⁰-substituted or unsubstituted alkyl, R³⁰-substituted or unsubstitutedheteroalkyl, R³⁰-substituted or unsubstituted cycloalkyl, R³⁰substituted or unsubstituted heterocycloalkyl, R³⁰-substituted orunsubstituted aryl, or R³⁰-substituted or unsubstituted heteroaryl.

R³⁰ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R³¹-substituted or unsubstituted alkyl, R³¹-substituted or unsubstitutedheteroalkyl, R³¹-substituted or unsubstituted cycloalkyl,R³¹-substituted or unsubstituted heterocycloalkyl, R³¹-substituted orunsubstituted aryl, or R³¹-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(7A) isindependently hydrogen, R^(38A)-substituted or unsubstituted alkyl,R^(38A)-substituted or unsubstituted heteroalkyl, R^(38A)-substituted orunsubstituted cycloalkyl, R^(38A)-substituted or unsubstitutedheterocycloalkyl, R^(38A)-substituted or unsubstituted aryl, orR^(38A)-substituted or unsubstituted membered heteroaryl.

R^(38A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(39A)-substituted or unsubstituted alkyl, R^(39A)-substituted orunsubstituted heteroalkyl, R^(39A)-substituted or unsubstitutedcycloalkyl, R^(39A)-substituted or unsubstituted heterocycloalkyl,R^(39A)-substituted or unsubstituted aryl, or R^(39A)-substituted orunsubstituted heteroaryl.

R^(39A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(40A)-substituted or unsubstituted alkyl, R^(40A)-substituted orunsubstituted heteroalkyl, R^(40A)-substituted or unsubstitutedcycloalkyl, R^(40A)-substituted or unsubstituted heterocycloalkyl,R^(40A)-substituted or unsubstituted aryl, or R^(40A)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(7B) isindependently hydrogen, R^(38B)-substituted or unsubstituted alkyl,R^(38B)-substituted or unsubstituted heteroalkyl, R^(38B)-substituted orunsubstituted cycloalkyl, R^(38B)-substituted or unsubstitutedheterocycloalkyl, R^(38B)-substituted or unsubstituted aryl, orR^(38B)-substituted or unsubstituted membered heteroaryl.

R^(38B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(39B)-substituted or unsubstituted alkyl, R^(39B)-substituted orunsubstituted heteroalkyl, R^(39B)-substituted or unsubstitutedcycloalkyl, R^(39B)-substituted or unsubstituted heterocycloalkyl,R^(39B)-substituted or unsubstituted aryl, or R^(39B)-substituted orunsubstituted heteroaryl.

R^(39B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(40B)-substituted or unsubstituted alkyl, R^(40B)-substituted orunsubstituted heteroalkyl, R^(40B)-substituted or unsubstitutedcycloalkyl, R^(40B)-substituted or unsubstituted heterocycloalkyl,R^(40B)-substituted or unsubstituted aryl, or R^(40B)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(8A) isindependently hydrogen, R^(41A)-substituted or unsubstituted alkyl,R^(41A)-substituted or unsubstituted heteroalkyl, R^(41A)-substituted orunsubstituted cycloalkyl, R^(41A)-substituted or unsubstitutedheterocycloalkyl, R^(41A)-substituted or unsubstituted aryl, orR^(41A)-substituted or unsubstituted heteroaryl.

R^(41A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(42A)-substituted or unsubstituted alkyl, R^(42A)-substituted orunsubstituted heteroalkyl, R^(42A)-substituted or unsubstitutedcycloalkyl, R^(42A)-substituted or unsubstituted heterocycloalkyl,R^(42A)-substituted or unsubstituted aryl, or R^(42A)-substituted orunsubstituted heteroaryl.

R^(42A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(43A)-substituted or unsubstituted alkyl, R^(43A)-substituted orunsubstituted heteroalkyl, R^(43A)-substituted or unsubstitutedcycloalkyl, R^(43A)-substituted or unsubstituted heterocycloalkyl,R^(43A)-substituted or unsubstituted aryl, or R^(43A)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(8B) isindependently hydrogen, R^(41B)-substituted or unsubstituted alkyl,R^(41B)-substituted or unsubstituted heteroalkyl, R^(41B)-substituted orunsubstituted cycloalkyl, R^(41B)-substituted or unsubstitutedheterocycloalkyl, R^(41B)-substituted or unsubstituted aryl, orR^(41B)-substituted or unsubstituted heteroaryl.

R^(41B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(42B)-substituted or unsubstituted alkyl, R^(42B)-substituted orunsubstituted heteroalkyl, R^(42B)-substituted or unsubstitutedcycloalkyl, R^(42B)-substituted or unsubstituted heterocycloalkyl,R^(42B)-substituted or unsubstituted aryl, or R^(42B)-substituted orunsubstituted heteroaryl.

R^(42B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(43B)-substituted or unsubstituted alkyl, R^(43B)-substituted orunsubstituted heteroalkyl, R^(43B)-substituted or unsubstitutedcycloalkyl, R^(43B)-substituted or unsubstituted heterocycloalkyl,R^(43B)-substituted or unsubstituted aryl, or R^(43B)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(9A) isindependently hydrogen, R^(44A)-substituted or unsubstituted alkyl,R^(44A)-substituted or unsubstituted heteroalkyl, R^(44A)-substituted orunsubstituted cycloalkyl, R^(44A)-substituted or unsubstitutedheterocycloalkyl, R^(44A)-substituted or unsubstituted aryl, orR^(44A)-substituted or unsubstituted heteroaryl.

R^(44A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(45A)-substituted or unsubstituted alkyl, R^(45A)-substituted orunsubstituted heteroalkyl, R^(45A)-substituted or unsubstitutedcycloalkyl, R^(45A)-substituted or unsubstituted heterocycloalkyl,R^(45A)-substituted or unsubstituted aryl, or R^(45A)-substituted orunsubstituted heteroaryl.

R^(45A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(46A)-substituted or unsubstituted alkyl, R^(46A)-substituted orunsubstituted heteroalkyl, R^(46A)-substituted or unsubstitutedcycloalkyl, R^(46A)-substituted or unsubstituted heterocycloalkyl,R^(46A)-substituted or unsubstituted aryl, or R^(46A)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(9B) isindependently hydrogen, R^(44B)-substituted or unsubstituted alkyl,R^(44B)-substituted or unsubstituted heteroalkyl, R^(44B)-substituted orunsubstituted cycloalkyl, R^(44B)-substituted or unsubstitutedheterocycloalkyl, R^(44B)-substituted or unsubstituted aryl, orR^(44B)-substituted or unsubstituted heteroaryl.

R^(44B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(45B)-substituted or unsubstituted alkyl, R^(45B)-substituted orunsubstituted heteroalkyl, R^(45B)-substituted or unsubstitutedcycloalkyl, R^(45B)-substituted or unsubstituted heterocycloalkyl,R^(45B)-substituted or unsubstituted aryl, or R^(45B)-substituted orunsubstituted heteroaryl.

R^(45B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(46B)-substituted or unsubstituted alkyl, R^(46B)-substituted orunsubstituted heteroalkyl, R^(46B)-substituted or unsubstitutedcycloalkyl, R^(46B)-substituted or unsubstituted heterocycloalkyl,R^(46B)-substituted or unsubstituted aryl, or R^(46B)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(10A) isindependently hydrogen, R^(47A)-substituted or unsubstituted alkyl,R^(47A)-substituted or unsubstituted heteroalkyl, R^(47A)-substituted orunsubstituted cycloalkyl, R^(47A)-substituted or unsubstitutedheterocycloalkyl, R^(47A)-substituted or unsubstituted aryl, orR^(47A)-substituted or unsubstituted heteroaryl.

R^(47A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(48A)-substituted or unsubstituted alkyl, R^(48A)-substituted orunsubstituted heteroalkyl, R^(48A)-substituted or unsubstitutedcycloalkyl, R^(48A)-substituted or unsubstituted heterocycloalkyl,R^(48A)-substituted or unsubstituted aryl, or R^(48A)-substituted orunsubstituted heteroaryl.

R^(48A) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(49A)-substituted or unsubstituted alkyl, R^(49A)-substituted orunsubstituted heteroalkyl, R^(49A)-substituted or unsubstitutedcycloalkyl, R^(49A)-substituted or unsubstituted heterocycloalkyl,R^(49A)-substituted or unsubstituted aryl, or R^(49A)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R^(10B) isindependently hydrogen, R^(47B)-substituted or unsubstituted alkyl,R^(47B)-substituted or unsubstituted heteroalkyl, R^(47B)-substituted orunsubstituted cycloalkyl, R^(47B)-substituted or unsubstitutedheterocycloalkyl, R^(47B)-substituted or unsubstituted aryl, orR^(47B)-substituted or unsubstituted heteroaryl.

R^(47B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(48B)-substituted or unsubstituted alkyl, R^(48B)-substituted orunsubstituted heteroalkyl, R^(48B)-substituted or unsubstitutedcycloalkyl, R^(48B)-substituted or unsubstituted heterocycloalkyl,R^(48B)-substituted or unsubstituted aryl, or R^(48B)-substituted orunsubstituted heteroaryl.

R^(48B) is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R^(49B)-substituted or unsubstituted alkyl, R^(49B)-substituted orunsubstituted heteroalkyl, R^(49B)-substituted or unsubstitutedcycloalkyl, R^(49B)-substituted or unsubstituted heterocycloalkyl,R^(49B)-substituted or unsubstituted aryl, or R^(49B)-substituted orunsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R¹¹ isindependently hydrogen, R⁵⁰-substituted or unsubstituted alkyl,R⁵⁰-substituted or unsubstituted heteroalkyl, R⁵⁰-substituted orunsubstituted cycloalkyl, R⁵⁰-substituted or unsubstitutedheterocycloalkyl, R⁵⁰-substituted or unsubstituted aryl, orR⁵⁰-substituted or unsubstituted heteroaryl.

R⁵⁰ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁵¹-substituted or unsubstituted alkyl, R⁵¹-substituted or unsubstitutedheteroalkyl, R⁵¹-substituted or unsubstituted cycloalkyl, R⁵¹substitutedor unsubstituted heterocycloalkyl, R⁵¹-substituted or unsubstitutedaryl, or R⁵¹-substituted or unsubstituted heteroaryl.

R⁵¹ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁵²-substituted or unsubstituted alkyl, R⁵²-substituted or unsubstitutedheteroalkyl, R⁵²-substituted or unsubstituted cycloalkyl,R⁵²-substituted or unsubstituted heterocycloalkyl, R⁵²-substituted orunsubstituted aryl, or R⁵²-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R¹² isindependently hydrogen, R⁵³-substituted or unsubstituted alkyl,R⁵³-substituted or unsubstituted heteroalkyl, R⁵³-substituted orunsubstituted cycloalkyl, R⁵³-substituted or unsubstitutedheterocycloalkyl, R⁵³-substituted or unsubstituted aryl, orR⁵³-substituted or unsubstituted heteroaryl.

R⁵³ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁵⁴-substituted or unsubstituted alkyl, R⁵⁴-substituted or unsubstitutedheteroalkyl, R⁵⁴-substituted or unsubstituted cycloalkyl, R⁵⁴substitutedor unsubstituted heterocycloalkyl, R⁵⁴-substituted or unsubstitutedaryl, or R⁵⁴-substituted or unsubstituted heteroaryl.

R⁵⁴ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁵⁵-substituted or unsubstituted alkyl, R⁵⁵-substituted or unsubstitutedheteroalkyl, R⁵⁵-substituted or unsubstituted cycloalkyl,R⁵⁵-substituted or unsubstituted heterocycloalkyl, R⁵⁵-substituted orunsubstituted aryl, or R⁵⁵-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R¹³ isindependently hydrogen, R⁵⁶-substituted or unsubstituted alkyl,R⁵⁶-substituted or unsubstituted heteroalkyl, R⁵⁶-substituted orunsubstituted cycloalkyl, R⁵⁶-substituted or unsubstitutedheterocycloalkyl, R⁵⁶-substituted or unsubstituted aryl, orR⁵⁶-substituted or unsubstituted heteroaryl.

R⁵⁶ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁵⁷-substituted or unsubstituted alkyl, R⁵⁷-substituted or unsubstitutedheteroalkyl, R⁵⁷-substituted or unsubstituted cycloalkyl, R⁵⁷substitutedor unsubstituted heterocycloalkyl, R⁵⁷-substituted or unsubstitutedaryl, or R⁵⁷-substituted or unsubstituted heteroaryl.

R⁵⁷ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁵⁸-substituted or unsubstituted alkyl, R⁵⁸-substituted or unsubstitutedheteroalkyl, R⁵⁸-substituted or unsubstituted cycloalkyl,R⁵⁸-substituted or unsubstituted heterocycloalkyl, R⁵⁸-substituted orunsubstituted aryl, or R⁵⁸-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R¹⁴ isindependently hydrogen, R⁵⁹-substituted or unsubstituted alkyl,R⁵⁹-substituted or unsubstituted heteroalkyl, R⁵⁹-substituted orunsubstituted cycloalkyl, R⁵⁹-substituted or unsubstitutedheterocycloalkyl, R⁵⁹-substituted or unsubstituted aryl, orR⁵⁹-substituted or unsubstituted heteroaryl.

R⁵⁹ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁶⁰-substituted or unsubstituted alkyl, R⁶⁰-substituted or unsubstitutedheteroalkyl, R⁶⁰-substituted or unsubstituted cycloalkyl,R⁶⁰-substituted or unsubstituted heterocycloalkyl, R⁶⁰-substituted orunsubstituted aryl, or R⁶⁰-substituted or unsubstituted heteroaryl.

R⁶⁰ independently is halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁶¹-substituted or unsubstituted alkyl, R⁶¹-substituted or unsubstitutedheteroalkyl, R⁶¹-substituted or unsubstituted cycloalkyl,R⁶¹-substituted or unsubstituted heterocycloalkyl, R⁶¹-substituted orunsubstituted aryl, or R⁶¹-substituted or unsubstituted heteroaryl.

In a further embodiment of the compounds provided herein, R¹⁵ isindependently hydrogen, R⁶²-substituted or unsubstituted alkyl,R⁶²-substituted or unsubstituted heteroalkyl, R⁶²-substituted orunsubstituted cycloalkyl, R⁶²-substituted or unsubstitutedheterocycloalkyl, R⁶²-substituted or unsubstituted aryl, orR⁶²-substituted or unsubstituted heteroaryl.

R⁶² is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁶³-substituted or unsubstituted alkyl, R⁶³-substituted or unsubstitutedheteroalkyl, R⁶³-substituted or unsubstituted cycloalkyl,R⁶³-substituted or unsubstituted heterocycloalkyl, R⁶³-substituted orunsubstituted aryl, or R⁶³-substituted or unsubstituted heteroaryl.

R⁶³ independently is halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁶⁴-substituted or unsubstituted alkyl, R⁶⁴-substituted or unsubstitutedheteroalkyl, R⁶⁴-substituted or unsubstituted cycloalkyl,R⁶⁴-substituted or unsubstituted heterocycloalkyl, R⁶⁴-substituted orunsubstituted aryl, or R⁶⁴-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R¹⁶ isindependently hydrogen, R⁶⁵-substituted or unsubstituted alkyl,R⁶⁵-substituted or unsubstituted heteroalkyl, R⁶⁵-substituted orunsubstituted cycloalkyl, R⁶⁵-substituted or unsubstitutedheterocycloalkyl, R⁶⁵-substituted or unsubstituted aryl, orR⁶⁵-substituted or unsubstituted heteroaryl.

R⁶⁵ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁶⁶-substituted or unsubstituted alkyl, R⁶⁶-substituted or unsubstitutedheteroalkyl, R⁶⁶-substituted or unsubstituted cycloalkyl,R⁶⁶-substituted or unsubstituted heterocycloalkyl, R⁶⁶-substituted orunsubstituted aryl, or R⁶⁶-substituted or unsubstituted heteroaryl.

R⁶⁶ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁶⁷-substituted or unsubstituted alkyl, R⁶⁷-substituted or unsubstitutedheteroalkyl, R⁶⁷-substituted or unsubstituted cycloalkyl,R⁶⁷-substituted or unsubstituted heterocycloalkyl, R⁶⁷-substituted orunsubstituted aryl, or R⁶⁷-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R¹⁷ isindependently hydrogen, R⁶⁸-substituted or unsubstituted alkyl,R⁶⁸-substituted or unsubstituted heteroalkyl, R⁶⁸-substituted orunsubstituted cycloalkyl, R⁶⁸-substituted or unsubstitutedheterocycloalkyl, R⁶⁸-substituted or unsubstituted aryl, orR⁶⁸-substituted or unsubstituted heteroaryl.

R⁶⁸ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁶⁹-substituted or unsubstituted alkyl, R⁶⁹-substituted or unsubstitutedheteroalkyl, R⁶⁹-substituted or unsubstituted cycloalkyl,R⁶⁹-substituted or unsubstituted heterocycloalkyl, R⁶⁹-substituted orunsubstituted aryl, or R⁶⁹-substituted or unsubstituted heteroaryl.

R⁶⁹ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁷⁰-substituted or unsubstituted alkyl, R⁷⁰-substituted or unsubstitutedheteroalkyl, R⁷⁰-substituted or unsubstituted cycloalkyl,R⁷⁰-substituted or unsubstituted heterocycloalkyl, R⁷⁰-substituted orunsubstituted aryl, or R⁷⁰-substituted or unsubstituted heteroaryl.

In some embodiments of the compounds provided herein, R¹⁸ isindependently hydrogen, R⁷¹-substituted or unsubstituted alkyl,R⁷¹-substituted or unsubstituted heteroalkyl, R⁷¹-substituted orunsubstituted cycloalkyl, R⁷¹-substituted or unsubstitutedheterocycloalkyl, R⁷¹-substituted or unsubstituted aryl, orR⁷¹-substituted or unsubstituted heteroaryl.

R⁷¹ is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁷²-substituted or unsubstituted alkyl, R⁷²-substituted or unsubstitutedheteroalkyl, R⁷²-substituted or unsubstituted cycloalkyl, R⁷²substituted or unsubstituted heterocycloalkyl, R⁷²-substituted orunsubstituted aryl, or R⁷²-substituted or unsubstituted heteroaryl.

R⁷² is independently halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,R⁷³-substituted or unsubstituted alkyl, R⁷³-substituted or unsubstitutedheteroalkyl, R⁷³-substituted or unsubstituted cycloalkyl,R⁷³-substituted or unsubstituted heterocycloalkyl, R⁷³-substituted orunsubstituted aryl, or R⁷³-substituted or unsubstituted heteroaryl.

R^(22A), R^(22B), R^(25A), R^(25B), R²⁸, R³¹, R^(40A), R^(40B), R^(43A),R^(43B), R^(46A), R^(46B), R^(49A), R^(49B), R⁵², R⁵⁵, R⁵⁸, R⁶¹, R⁶⁴,R⁶⁷, R⁷⁰, and R⁷³, are independently hydrogen, halogen, —CF₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC═(O)NHNH₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, or unsubstituted heteroaryl.

In some embodiments, the compound is any one of the compounds in theExamples section below.

In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is an eEF-2K modulator. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is an eEF-2K inhibitor. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is an eEF-2K activator. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is an eEF-2 modulator. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is an eEF-2 inhibitor. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is an eEF-2 activator. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR_(1B) modulator.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR_(1B) inhibitor.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR_(1B) activator.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR_(1D) modulator.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR_(1D) inhibitor.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR_(1D) activator.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR modulator. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR inhibitor. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound is a 5-HTR activator. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), OHO, (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound reduces the effectiveamount necessary to treat a disease. In some embodiments of the compounddescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe),(If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), thecompound reduces the effective amount of a second agent (e g inhibitor,modulator, compound, drug, chemotherapeutic agent) necessary to treat adisease. In some embodiments of the compound described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments), the compound is an mTORinhibitor. In some embodiments of the compound described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), OHO, (Ig), (IIIg),(Ih), (IIIh), including embodiments), the compound is an Akt inhibitor.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound inhibits cancer cellproliferation. In some embodiments of the compound described herein(e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa),(Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc),(Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (Hie), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments), the compound inhibitscancer cell survival. In some embodiments of the compound describedherein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa),(IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc),(IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If),(IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), the compoundinhibits cancer cell migration/invasion. In some embodiments of thecompound described herein (e.g. Formula (I), (II), (III), (IV), (V),(Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments), the compound inhibits cancer cell cell cycle progression.In some embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound inhibits angiogenesis. Insome embodiments of the compound described herein (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih),(IIIh), including embodiments), the compound inhibits the level of c-mycactivity in a cell. In some embodiments of the compound as describedherein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa),(IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc),(IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If),(IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), the compoundinhibits the level of cyclin-D1 activity in a cell. In some embodimentsof the compound as described herein (e.g. Formula (I), (II), (III),(IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb),(Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd),(Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments), the compound inhibits the level of Bcl-2 activity in acell. In some embodiments of the compound as described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments), the compound inhibits thelevel of VEGF activity. In some embodiments of the compound as describedherein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa),(IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc),(IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If),(IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), the compoundinhibits the level of HIF1alpha activity in a cell. In some embodimentsof the compound as described herein (e.g. Formula (I), (II), (III),(IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb),(Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd),(Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments), the compound inhibits the level of c-Src activity in acell. In some embodiments of the compound as described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments), the compound inhibits thelevel of FAK activity in a cell. In some embodiments of the compound asdescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (Hie),(If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), thecompound inhibits the level of Paxillin activity in a cell. In someembodiments of the compound as described herein (e.g. Formula (I), (II),(III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb),(IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId),(IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh),including embodiments), the compound inhibits the level of IGF-1Ractivity in a cell. In some embodiments of the compound as describedherein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa),(IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc),(IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If),(IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), the compoundinhibits the level of Akt activity in a cell. In some embodiments of thecompound as described herein (e.g. Formula (I), (II), (III), (IV), (V),(Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (IIIe), (If), OHO, (Ig), (IIIg), (Ih), (IIIh), includingembodiments), the compound inhibits the level of mTOR activity in acell. In some embodiments of the compound as described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments), the compound inhibits thelevel of NF-κB activity in a cell. In some embodiments of the compoundas described herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia),(IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic),(IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie),(IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments), the compound inhibits the level of ERK1 activity in acell. In some embodiments of the compound as described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (Hie), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments), the compound inhibits thelevel of ERK2 activity in a cell. In some embodiments of the compound asdescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe),(If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), thecompound inhibits the level of MAPK activity in a cell.

In another aspect, one or more hydrogens of the compounds providedherein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa),(IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc),(IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If),(IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments), may besubstituted with an —F. The terms “compounds described herein”,“compounds as described herein”, “compounds provided herein”,“compositions of the present invention”, and “compounds of the presentinvention” may be used interchangeably.

In some embodiments, a compound as described herein may include multipleinstances of R³, R⁴, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, X³, X⁴, u,k, m, n, q, r, t, and/or other variables. In such embodiments, eachvariable may optional be different and be appropriately labeled todistinguish each group for greater clarity. For example, where each R³,R⁴, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, X³, X⁴, u, k, m, n, q, r,and t is different, they may be referred to, for example, as R^(3c),R^(3d), R^(3e), R^(3f), R^(3g), R^(3h), R^(3i), R^(4c), R^(4d), R^(4e),R^(4f), R^(4g), R^(4h), R^(4i), R^(11c), R^(11d), R^(11e), R^(11f),R^(11g), R^(11h), R^(11i), R^(12c), R^(12d), R^(12e), R^(12f), R^(12g),R^(12h), R^(12i), R^(13c), R^(13d), R^(13e), R^(13f), R^(13g), R^(13h),R^(13i), R^(14c), R^(14d), R^(14e), R^(14f), R^(14g), R^(14h), R^(14i),R^(15c), R^(15d), R^(15e), R^(15f), R^(15g), R^(15h), R^(15i), R^(16c),R^(16d), R^(16e), R^(16f), R^(16g), R^(16h), R^(16i), R^(17c), R^(17d),R^(17e), R^(17f), R^(17g), R^(17h), R^(17i), R^(18c), R^(18d), R^(18e),R^(18f), R^(18g), R^(18h), R^(18i), X^(3c), X^(3d), X^(3e), X^(3f),X^(3g), X^(3h), X^(3i), X^(4c), X^(4d), X^(4e), X^(4f), X^(4g), X^(4h),X^(4i), u^(C), u^(d), u^(e), u^(f), u^(g), u^(h), u^(i), k^(c), k^(d),k^(e), k^(f), k^(g), k^(h), k^(i), m^(c), m^(d), m^(e), m^(f), m^(g),m^(h), m^(i), n^(c), n^(d), n^(e), n^(f), n^(g), n^(h), n^(i), q^(c),q^(d), q^(e), q^(f), q^(g), q^(h), q^(i), r^(c), r^(d), r^(e), r^(f),r^(g), r^(h), r^(i), t^(c), t^(d), t^(e), t^(f), t^(g), t^(h), t^(i),respectively, wherein the definition of R³ is assumed by R^(3c), R^(3d),R^(3e), R^(3f), R^(3g), R^(3h), R^(3i), the definition of R⁴ is assumedby R^(4c), R^(4d), R^(4e), R^(4f), R^(4g), R^(4h), R^(4i), thedefinition of R¹¹ is assumed by R^(11c), R^(11d), R^(11e), R^(11f),R^(11g), R^(11h), R^(11i), the definition of R¹² is assumed by R^(12c),R^(12d), R^(12e), R^(12f), R^(12g), R^(12h), R^(12i), the definition ofR¹³ is assumed by R^(13c), R^(13d), R^(13e), R^(13f), R^(13g), R^(13h),R^(13i), the definition of R¹⁴ is assumed by R^(14c), R^(14d), R^(14e),R^(14f), R^(14g), R^(14h), R^(14i), the definition of R¹⁵ is assumed byR^(15c), R^(15d), R^(15e), R^(15f), R^(15g), R^(15h), R^(15i), thedefinition of R¹⁶ is assumed by R^(16c), R^(16d), R^(16e), R^(16f),R^(16g), R^(16h), the definition of R¹⁷ is assumed by R^(17c), R^(17d),R^(17e), R^(17f), R^(17g), R^(17h), R^(17i), the definition of R¹⁸ isassumed by R^(18c), R^(18d), Re, R^(18f), R^(18g), R^(18h), R^(18i), thedefinition of X³ is assumed by X^(3c), X^(3d), X^(3e), X^(3f), X^(3g),X^(3h), X^(3i), the definition of X⁴ is assumed by X^(4c), X^(4d),X^(4e), X^(4f), X^(4g), X^(4h), X^(4i), the definition of u is assumedby u^(c), u^(d), u^(e), u^(f), u^(g), u^(h), u^(i), the definition of kis assumed by k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), k^(i), thedefinition of m is assumed by m^(c), m^(d), m^(e), m^(f), m^(g), m^(h),m^(i), the definition of n is assumed by n^(c), n^(d), n^(e), n^(f),n^(g), n^(h), n^(i), the definition of q is assumed by q^(c), q^(d),q^(e), q^(f), q^(g), q^(h), q^(i), the definition of r is assumed byr^(c), r^(d), r^(e), r^(f), r^(g), r^(h), r^(i), the definition oft isassumed by t^(c), t^(d), t^(e), t^(f), t^(g), t^(h), t^(i).

The variables used within a definition of R³, R⁴, R¹¹, R¹², R¹³, R¹⁴,R¹⁵, R¹⁶, R¹⁷, R¹⁸, X³, X⁴, u, k, m, n, q, r, t, and/or other variablesthat appear at multiple instances and are different may similarly beappropriately labeled to distinguish each group for greater clarity. Forexample, in some embodiments, the definition of R^(3c) may employ thesymbols R^(4c), R^(11c), R^(12c), R^(13c), R^(14c), X^(3c), u^(c),k^(c), m^(c), and n^(c) which assume the definitions of R⁴, R¹¹, R¹²,R¹³, R¹⁴, X³, u, k, m, and n, respectively. In some embodiments, thedefinition of R^(4c) may employ the symbols R^(15c), R^(16c), R^(17c),R^(18c), X^(4c), q^(c), r^(c), and t^(c) which assume the definitions ofR¹⁵, R¹⁶, R¹⁷, R¹⁸, X⁴, q, r, and t, respectively. In some embodiments,the definition of R^(3d) may employ the symbols R^(4d), R^(11d),R^(12d), R^(13d), R^(14d), X^(3d), u^(d), k^(d), m^(d), and n^(d) whichassume the definitions of R⁴, R¹¹, R¹², R¹³, R¹⁴, X³, u, k, m, and n,respectively. In some embodiments, the definition of R^(4d) may employthe symbols R^(15d), R^(16d), R^(17d), R^(18d), X^(4d), q^(d), r^(d),and t^(d) which assume the definitions of R¹⁵, R¹⁶, R¹⁷, R¹⁸, X⁴, q, r,and t, respectively. In some embodiments, the definition of R^(3e) mayemploy the symbols R^(4e), R^(11e), R^(12e), R^(13e), R^(14e), X^(3e),u^(e), k^(e), m^(e), and n^(e) which assume the definitions of R⁴, R¹¹,R¹², R¹³, R¹⁴, X³, u, k, m, and n, respectively. In some embodiments,the definition of R^(4e) may employ the symbols R^(15e), R^(16e),R^(17e), R^(18e), X^(4e), q^(e), r^(e), and t^(e) which assume thedefinitions of R¹⁵, R¹⁶, R¹⁷, R¹⁸, X⁴, q, r, and t, respectively. Insome embodiments, the definition of R^(3f) may employ the symbolsR^(4f), R^(11f), R^(12f), R^(13f), R^(14f), X^(3f), u^(f), k^(f), m^(f),and n^(f) which assume the definitions of R⁴, R¹¹, R¹², R¹³, R¹⁴, X³, u,k, m, and n, respectively. In some embodiments, the definition of R^(4f)may employ the symbols R^(15f), R^(16f), R^(18f), X^(4f), q^(f), r^(f),and t^(f) which assume the definitions of R¹⁵, R¹⁶, R¹⁷, R¹⁸, X⁴, q, r,and t, respectively. In some embodiments, the definition of R^(3g) mayemploy the symbols R^(4g), R^(11g), R^(12g), R^(13g), R^(14g), X^(3g),u^(g), k^(g), m^(g), and n^(g) which assume the definitions of R⁴, R¹¹,R¹², R¹³, R¹⁴, X³, u, k, m, and n, respectively. In some embodiments,the definition of R^(4g) may employ the symbols R^(15g), R^(16g),R^(17g), R^(18g), X^(4g), q^(g), r^(g), and t^(g) which assume thedefinitions of R¹⁵, R¹⁶, R¹⁷, R¹⁸, X⁴, q, r, and t, respectively. Insome embodiments, the definition of R^(3h) may employ the symbolsR^(4h), R^(11h), R^(12h), R^(13h), R^(14h), X^(3h), u^(h), k^(h), m^(h),and n^(h) which assume the definitions of R⁴, R¹¹, R¹², R¹³, R¹⁴, X³, u,k, m, and n, respectively. In some embodiments, the definition of R^(4h)may employ the symbols R^(15h), R^(16h), R^(17h), R^(18h), X^(4h),q^(h), r^(h), and t^(h) which assume the definitions of R¹⁵, R¹⁶, R¹⁷,R¹⁸, X⁴, q, r, and t, respectively. In some embodiments, the definitionof R^(3i) may employ the symbols R^(4i), R^(11i), R^(12i), R^(13i),R^(14i), X^(3i), u^(i), k^(i), m^(i), and n^(i) which assume thedefinitions of R⁴, R¹¹, R¹², R¹³, R¹⁴, X³, u, k, m, and n, respectively.In some embodiments, the definition of R^(4i) may employ the symbolsR^(15i), R^(16i), R^(17i), R^(18i), X^(4i), q^(i), r^(i), and t^(i)which assume the definitions of R¹⁵, R¹⁶, R¹⁷, R¹⁸, X⁴, q, r, and t,respectively.

Pharmaceutical Compositions and Methods of Treatment

In a second aspect, a pharmaceutical composition is provided thatincludes a pharmaceutically acceptable excipient and a compound asdescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (Hie),(If), (IIIf), (Ig), (Ing), (Ih), (IIIh), including embodiments).

In another aspect, a pharmaceutical composition is provided thatincludes a pharmaceutically acceptable excipient and an anti-eEF-2Kinhibitory nucleic acid, anti-5-HTR_(1B) inhibitory nucleic acid,anti-5-HTR_(1D) inhibitory nucleic acid, or an anti-5-HTR inhibitorynucleic acid. In some embodiments, the pharmaceutical compositionincludes a pharmaceutically acceptable excipient and an inhibitorynucleic acid, the inhibitory nucleic acid is an anti-eEF-2K siRNA,anti-5-HTR_(1B) siRNA, anti-5-HTR_(1D) siRNA, or anti-5-HTR siRNA. Insome embodiments of the pharmaceutical composition, the inhibitorynucleic acid is an anti-eEF-2K antisense nucleic acid, anti-5-HTR_(1B)antisense nucleic acid, anti-5-HTR_(1D) antisense nucleic acid, or ananti-5-HTR antisense nucleic acid. In some embodiments of thepharmaceutical composition that includes a pharmaceutically acceptableexcipient and an antisense nucleic acid, the antisense nucleic acid isan anti-eEF-2K siRNA, anti-5-HTR_(1B) siRNA, anti-5-HTR_(1D) siRNA, oranti-5-HTR siRNA.

In some embodiments of the pharmaceutical compositions provided herein,the pharmaceutical composition includes a pharmaceutically acceptableexcipient, a liposome, and a compound or inhibitory (e.g. antisense)nucleic acid as described herein (e.g. Formula (I), (II), (III), (IV),(V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (Hie), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments). In some embodiments of the pharmaceutical composition, theliposome includes dioleoyl-sn-glycero-3-phosphocholine. In someembodiments of the pharmaceutical composition, the liposome includesdimyristoyl-phosphatidylcholine. In some embodiments of thepharmaceutical composition, the compound or inhibitory (e.g. antisense)nucleic acid (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe),(If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments) isinside the liposome. In some embodiments of the pharmaceuticalcomposition, the compound or inhibitory (e.g. antisense) nucleic acid(e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa),(Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc),(Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf),(Ig), (IIIg), (Ih), (IIIh), including embodiments) is part of theliposome. In some embodiments of the pharmaceutical composition, theliposome includes a targeting moiety. In some embodiments of thepharmaceutical composition, the targeting moiety is folate. A targetingmoiety that is part of a liposome, as the term used herein, refers to amoiety that directs the liposome to a particular location (e.g. tissuetype, cell type, organ). In some embodiments, a targeting moiety is acompound. In some embodiments, a targeting moiety is a protein. In someembodiments, a targeting moiety is a nucleic acid. In some embodiments,a targeting moiety binds a cell. In some embodiments, a targeting moietybinds a protein. In some embodiments, a targeting moiety binds a proteinon a cell. In some embodiments, a targeting moiety binds a protein in acell. In some embodiments, a targeting moiety binds a cancer cell.

In a third aspect, a method of treating a disease in a patient in needof such treatment is provided. The method includes administering atherapeutically effective amount of a compound as described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments).

In another aspect, a method of treating a disease in a patient in needof such treatment is provided. The method includes administering atherapeutically effective amount of an anti-eEF-2K inhibitory (e.g.antisense) nucleic acid, anti-5-HTR_(1B) inhibitory (e.g. antisense)nucleic acid, anti-5-HTR_(1D) inhibitory (e.g. antisense) nucleic acid,or an anti-5-HTR inhibitory (e.g. antisense) nucleic acid. In someembodiments of the method of treating a disease in a patient in need ofsuch treatment that includes administering a therapeutically effectiveamount of an inhibitory (e.g. antisense) nucleic acid, the inhibitory(e.g. antisense) nucleic acid is an anti-eEF-2K siRNA, anti-5-HTR_(1B)siRNA, anti-5-HTR_(1D) siRNA, or anti-5-HTR siRNA.

In some embodiments of the method of treating a disease, the disease iscancer. In some embodiments of the method of treating a disease, thedisease is breast cancer. In some embodiments of the method of treatinga disease, the disease is ovarian cancer. In some embodiments of themethod of treating a disease, the disease is pancreatic cancer. In someembodiments of the method of treating a disease, the disease ismelanoma. In some embodiments of the method of treating a disease, thedisease is lung cancer. In some embodiments of the method of treating adisease, the disease is glioblastoma. In some embodiments of the methodof treating a disease, the disease is glioma. In some embodiments of themethod of treating a disease, the disease is bladder cancer. In someembodiments of the method of treating a disease, the disease is livercancer. In some embodiments of the method of treating a disease, thedisease is depression. In some embodiments of the method of treating adisease, the disease is metastatic cancer. In some embodiments of themethod of treating a disease, the disease is metastatic breast cancer.In some embodiments of the method of treating a disease, the disease ismetastatic ovarian cancer. In some embodiments of the method of treatinga disease, the disease is metastatic pancreatic cancer. In someembodiments of the method of treating a disease, the disease ismetastatic melanoma. In some embodiments of the method of treating adisease, the disease is metastatic lung cancer. In some embodiments ofthe method of treating a disease, the disease is metastaticglioblastoma. In some embodiments of the method of treating a disease,the disease is metastatic glioma. In some embodiments of the method oftreating a disease, the disease is metastatic bladder cancer. In someembodiments of the method of treating a disease, the disease ismetastatic liver cancer. In some embodiments of the method of treating adisease, the cancer has metastasized to a different location in the bodyfrom the primary tumor. In some embodiments of a method of treatingcancer, the cancer is associated with an increased level of activity ofeEF-2K. In some embodiments of a method of treating cancer, the canceris associated with an increased amount of eEF-2K. In some embodiments ofa method of treating cancer, the cancer is associated with an increasedlevel of activity of 5-HTR_(1B). In some embodiments of a method oftreating cancer, the cancer is associated with an increased amount of5-HTR_(1B). In some embodiments of a method of treating cancer, thecancer is associated with an increased level of activity of 5-HTR_(1D).In some embodiments of a method of treating cancer, the cancer isassociated with an increased amount of 5-HTR_(1D). In some embodimentsof a method of treating cancer, the cancer has failed to be treated by acompound not provided herein. In some embodiments of a method oftreating cancer, the cancer has failed to be treated by a compoundapproved for treating the cancer by a government regulator agency. Insome embodiments of the method of treating cancer, the disease is aprimary tumor. In some embodiments of the method of treating a disease,the disease is a hyperproliferative disorder. In some embodiments of themethod of treating a disease, the disease is estrogen receptor positivebreast cancer. In some embodiments of the method of treating a disease,the disease is estrogen receptor (ER) negative breast cancer. In someembodiments of the method of treating a disease, the disease istamoxifen resistant breast cancer. In some embodiments of the method oftreating a disease, the disease is HER2 negative breast cancer. In someembodiments of the method of treating a disease, the disease is HER2positive breast cancer. In some embodiments of the method of treating adisease, the disease is low grade (well differentiated) breast cancer.In some embodiments of the method of treating a disease, the disease isintermediate grade (moderately differentiated) breast cancer. In someembodiments of the method of treating a disease, the disease is highgrade (poorly differentiated) breast cancer. In some embodiments of themethod of treating a disease, the disease is stage 0 breast cancer. Insome embodiments of the method of treating a disease, the disease isstage I breast cancer. In some embodiments of the method of treating adisease, the disease is stage II breast cancer. In some embodiments ofthe method of treating a disease, the disease is stage III breastcancer. In some embodiments of the method of treating a disease, thedisease is stage IV breast cancer.

In some embodiments of the method of treating a disease, the compound orinhibitory (e.g. antisense) nucleic acid is co-administered with achemotherapeutic agent. The term “chemotherapeutic agent” is used inaccordance with its plain ordinary meaning and refers to a compositionor compound that is an antineoplastic drug (e.g. alkylating agents,antimetabolites, anthracyclines, alkaloids, topoisomerase inhibitors,select monoclonal antibodies, kinase inhibitors, tyrosine kinaseinhibitors, select cytotoxic antibiotics, taxanes, actinomycin,bleomycin, plicamycin, mitomycin, targeted cancer therapies). In someembodiments of the method of treating a disease, the compound orinhibitory (e.g. antisense) nucleic acid is co-administered with ananthracycline. In some embodiments of the method of treating a disease,the compound or inhibitory (e.g. antisense) nucleic acid isco-administered with doxorubicin (dox). In some embodiments of themethod of treating a disease, the compound or inhibitory (e.g.antisense) nucleic acid is co-administered with an alkylating agent(e.g. cisplatin, carboplatin, oxaliplatin, mechlorethamine,cyclophosphamide, chlorambucil, ifosfamide)). In some embodiments of themethod of treating a disease, the compound or inhibitory (e.g.antisense) nucleic acid is co-administered with an antimetabolite (e.g.azathioprine, mercaptopurine). In some embodiments of the method oftreating a disease, the compound or inhibitory (e.g. antisense) nucleicacid is co-administered with a vinca alkaloid (e.g. vincristine,vinblastine, vinorelbine, or vindesine). In some embodiments of themethod of treating a disease, the compound or inhibitory (e.g.antisense) nucleic acid is co-administered with a taxane (e.g.paclitaxel (Taxol) or docetaxel). In some embodiments of the method oftreating a disease, the compound or inhibitory (e.g. antisense) nucleicacid is co-administered with a topoisomerase inhibitor (e.g. irinotecan,topotecan, amsacrine, etoposide, etoposide phosphate, or teniposide). Insome embodiments of the method of treating a disease, the compound orinhibitory (e.g. antisense) nucleic acid is co-administered withactinomycin. In some embodiments of the method of treating a disease,the compound or inhibitory (e.g. antisense) nucleic acid isco-administered with an anthracycline (e.g. doxorubicin, daunorubicin,valrubicin, idarubicin, or epirubicin). In some embodiments of themethod of treating a disease, the compound or inhibitory (e.g.antisense) nucleic acid is co-administered with bleomycin. In someembodiments of the method of treating a disease, the compound orinhibitory (e.g. antisense) nucleic acid is co-administered withplicamycin. In some embodiments of the method of treating a disease, thecompound or inhibitory (e.g. antisense) nucleic acid is co-administeredwith mitomycin. In some embodiments of the method of treating a disease,the compound or inhibitory (e.g. antisense) nucleic acid isco-administered with radiation therapy. In some embodiments of themethod of treating a disease, the compound or inhibitory (e.g.antisense) nucleic acid is co-administered with hormonal therapy (e.g.aromatase inhibitors, gonadotropin-releasing hormone analogs, selectiveestrogen receptor modulators, progestins, or antiandrogens). In someembodiments of the method of treating a disease, the compound orinhibitory (e.g. antisense) nucleic acid is co-administered with anaromatase inhibitor (e.g. letrozole, anastrozole, or exemestane). Insome embodiments of the method of treating a disease, the compound orinhibitory (e.g. antisense) nucleic acid is co-administered withtamoxifen. In some embodiments of the method of treating a disease, thecompound or inhibitory (e.g. antisense) nucleic acid is co-administeredwith a gonadotropin-releasing hormone analog (e.g. leuprolide orgoserelin). In some embodiments of the method of treating a disease, thecompound or inhibitory (e.g. antisense) nucleic acid is co-administeredwith a selective estrogen receptor modulator (e.g. tamoxifen,raloxifene, toremifene, or fulvestrant). In some embodiments of themethod of treating a disease, the compound or inhibitory (e.g.antisense) nucleic acid is co-administered with an antiandrogen (e.g.flutamide or bicalutamide). In some embodiments of the method oftreating a disease, the compound or inhibitory (e.g. antisense) nucleicacid is co-administered with a progestin (e.g. megestrol ormedroxyprogesterone).

In some embodiments, a chemotherapeutic agent is an agent approved bythe FDA or similar regulatory agency of a country other than the USA,for treating cancer. Examples of chemotherapeutic agents include, butare not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors(e.g. XL518, CI-1040, PD035901, selumetinib/AZD6244,GSK1120212/trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901,U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylatingagents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan,melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogenmustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil,meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine,thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine, lomusitne, semustine, streptozocin), triazenes(decarbazine)), anti-metabolites (e.g., 5-azathioprine, leucovorin,capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folicacid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g.,vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g.,irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate,teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin,daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g.cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g.,mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazinederivative (e.g., procarbazine), adrenocortical suppressant (e.g.,mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide),antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g.,L-asparaginase), inhibitors of mitogen-activated protein kinasesignaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY-142886,SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002, Sykinhibitors, mTOR inhibitors, antibodies (e.g., rituxan), gossyphol,genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA),bryostatin, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL), 5-aza-2′-deoxycytidine, all trans retinoic acid, doxorubicin,vincristine, etoposide, gemcitabine, imatinib (Gleevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352,20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatinstimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin I1 (includingrecombinant interleukin II, or r1L.sub.2), interferon alfa-2a;interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferonbeta-1a; interferon gamma-lb; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride, agents that arrest cells in the G2-M phases and/ormodulate the formation or stability of microtubules, (e.g. Taxol™ (i.e.paclitaxel), Taxotere™, compounds comprising the taxane skeleton,Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128),Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829,Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010),Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, andSpongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 andNSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, EpothiloneC (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB,and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone BN-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B(i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F anddEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin(i.e. TZT-1027), LS-4559-P (Pharmacia, i.e. LS-4577), LS-4578(Pharmacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia),RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877(Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2(Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 andLU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis),AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko),IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto,i.e. AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e. AVE-8062,AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, TubulysinA, Canadensol, Centaureidin (i.e. NSC-106969), T-138067 (Tularik, i.e.T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e.DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas StateUniversity), Oncocidin A1 (i.e. BTO-956 and DIME), DDE-313 (ParkerHughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker HughesInstitute), SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU(Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-569), Narcosine(also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972(Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School ofMedicine, i.e. MF-191), TMPN (Arizona State University), Vanadoceneacetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine (i.e.NSC-698666), 3-IAABE (Cytoskeleton/Mt. Sinai School of Medicine),A-204197 (Abbott), T-607 (Tuiarik, i.e. T-900607), RPR-115781 (Aventis),Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin,lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin,Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica),Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A,TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (−)-Phenylahistin(i.e. NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica),Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A-289099 (Abbott),A-318315 (Abbott), HTI-286 (i.e. SPA-110, trifluoroacetate salt)(Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI),Resverastatin phosphate sodium, BPR-OY-007 (National Health ResearchInstitutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone),finasteride, aromatase inhibitors, gonadotropin-releasing hormoneagonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids(e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate,megestrol acetate, medroxyprogesterone acetate), estrogens (e.g.,diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen),androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen(e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guérin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™),erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, or the like.

In some embodiments of a method of treating cancer, the cancer isassociated with aberrant levels of eEF-2. In some embodiments of amethod of treating cancer, the cancer is associated with aberrant levelsof eEF-2 activity. In some embodiments of a method of treating cancer,the cancer is associated with aberrant levels of eEF-2K. In someembodiments of a method of treating cancer, the cancer is associatedwith aberrant levels of eEF-2K activity. In some embodiments of a methodof treating cancer, the cancer is associated with aberrant levels of5-HTR. In some embodiments of a method of treating cancer, the cancer isassociated with aberrant levels of 5-HTR activity. In some embodimentsof a method of treating cancer, the cancer is associated with aberrantlevels of 5-HTR_(1B). In some embodiments of a method of treatingcancer, the cancer is associated with aberrant levels of -HTR_(1B)activity. In some embodiments of a method of treating cancer, the canceris associated with aberrant levels of -HTR_(1D). In some embodiments ofa method of treating cancer, the cancer is associated with aberrantlevels of -HTR_(1D) activity.

In some embodiments of the method of treating a disease, the disease isdepression. In some embodiments of the method of treating a disease, thedisease is migraine headaches. In some embodiments of the method oftreating a disease, the disease is pain.

The pharmaceutical compositions include optical isomers, diastereomers,or pharmaceutically acceptable salts of the modulators disclosed herein.The compound included in the pharmaceutical composition may becovalently attached to a carrier moiety, as described above.Alternatively, the compound included in the pharmaceutical compositionis not covalently linked to a carrier moiety.

The compounds or inhibitory (e.g. antisense) nucleic acid of theinvention can be administered alone or can be coadministered to thepatient. Coadministration is meant to include simultaneous or sequentialadministration of the compounds or inhibitory (e.g. antisense) nucleicacids individually or in combination (more than one compound orinhibitory (e.g. antisense) nucleic acid or combination of both). Thus,the preparations can also be combined, when desired, with other activesubstances (e.g. to reduce metabolic degradation).

The compounds or inhibitory (e.g. antisense) nucleic acids of thepresent invention can be prepared and administered in a wide variety oforal, parenteral and topical dosage forms. Oral preparations includetablets, pills, powder, dragees, capsules, liquids, lozenges, cachets,gels, syrups, slurries, suspensions, etc., suitable for ingestion by thepatient. The compounds or inhibitory (e.g. antisense) nucleic acids ofthe present invention can also be administered by injection, that is,intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally. Also, the compounds or inhibitory(e.g. antisense) nucleic acids described herein can be administered byinhalation, for example, intranasally. Additionally, the compounds orinhibitory (e.g. antisense) nucleic acids of the present invention canbe administered transdermally. It is also envisioned that multipleroutes of administration (e.g., intramuscular, oral, transdermal) can beused to administer the compounds or inhibitory (e.g. antisense) nucleicacids of the invention. Accordingly, the present invention also providespharmaceutical compositions comprising a pharmaceutically acceptableexcipient and one or more compounds or inhibitory (e.g. antisense)nucleic acids of the invention.

For preparing pharmaceutical compositions from the compounds orinhibitory (e.g. antisense) nucleic acids of the present invention,pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier can beone or more substance, that may also act as diluents, flavoring agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier is a finely divided solid in a mixture with thefinely divided active component (e.g. a compound or inhibitory (e.g.antisense) nucleic acid provided herein). In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% to 70% ofthe active compound or inhibitory (e.g. antisense) nucleic acid.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; talc; pectin; dextrin; starch;tragacanth; a low melting wax; cocoa butter; carbohydrates; sugarsincluding, but not limited to, lactose, sucrose, mannitol, or sorbitol,starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins including, but not limited to, gelatin and collagen. Ifdesired, disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

When parenteral application is needed or desired, particularly suitableadmixtures for the compounds or inhibitory (e.g. antisense) nucleicacids of the invention are injectable, sterile solutions, preferablyoily or aqueous solutions, as well as suspensions, emulsions, orimplants, including suppositories. In particular, carriers forparenteral administration include aqueous solutions of dextrose, saline,pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,polyoxyethylene-block polymers, and the like. Ampules are convenientunit dosages. The compounds or inhibitory (e.g. antisense) nucleic acidsof the invention can also be incorporated into liposomes or administeredvia transdermal pumps or patches. Pharmaceutical admixtures suitable foruse in the present invention are well-known to those of skill in the artand are described, for example, in Pharmaceutical Sciences (17th Ed.,Mack Pub. Co., Easton, Pa.) and WO 96/05309, the teachings of both ofwhich are hereby incorporated by reference.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can contain a thickening agent, such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents can be added to provide apalatable oral preparation, such as glycerol, sorbitol or sucrose. Theseformulations can be preserved by the addition of an antioxidant such asascorbic acid. As an example of an injectable oil vehicle, see Minto, J.Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical formulationsof the invention can also be in the form of oil-in-water emulsions. Theoily phase can be a vegetable oil or a mineral oil, described above, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to1000 mg, most typically 10 mg to 500 mg, according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Some compounds or inhibitory (e.g. antisense) nucleic acids may havelimited solubility in water and therefore may require a surfactant orother appropriate co-solvent in the composition. Such co-solventsinclude: Polysorbate 20, 60 and 80; Pluronic F-68, F-84 and P-103;cyclodextrin; polyoxyl 35 castor oil; or other agents known to thoseskilled in the art. Such co-solvents are typically employed at a levelbetween about 0.01% and about 2% by weight.

Viscosity greater than that of simple aqueous solutions may be desirableto decrease variability in dispensing the formulations, to decreasephysical separation of components of a suspension or emulsion offormulation and/or otherwise to improve the formulation. Such viscositybuilding agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid andsalts thereof, combinations of the foregoing, and other agents known tothose skilled in the art. Such agents are typically employed at a levelbetween about 0.01% and about 2% by weight. Determination of acceptableamounts of any of the above adjuvants is readily ascertained by oneskilled in the art.

The compositions of the present invention may additionally includecomponents to provide sustained release and/or comfort. Such componentsinclude high molecular weight, anionic mucomimetic polymers, gellingpolysaccharides and finely-divided drug carrier substrates. Thesecomponents are discussed in greater detail in U.S. Pat. Nos. 4,911,920;5,403,841; 5,212,162; and 4,861,760. The entire contents of thesepatents are incorporated herein by reference in their entirety for allpurposes.

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. When administered in methods to treat a disease, suchcompositions will contain an amount of active ingredient effective toachieve the desired result, e.g., modulating the activity of a targetmolecule (e.g. a kinase or kinase(s); eEF-2K, eEF-2, 5-HTR_(1B),5-HTR_(1D), 5-HTR), and/or reducing, eliminating, or slowing theprogression of disease symptoms (e.g. cancer growth or metastasis,depression). Determination of a therapeutically effective amount of acompound or inhibitory (e.g. antisense) nucleic acid of the invention iswell within the capabilities of those skilled in the art, especially inlight of the detailed disclosure herein.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated (e.g. cancer, depression), kind of concurrent treatment,complications from the disease being treated or other health-relatedproblems. Other therapeutic regimens or agents can be used inconjunction with the methods and compounds or inhibitory (e.g.antisense) nucleic acids of Applicants' invention. Adjustment andmanipulation of established dosages (e.g., frequency and duration) arewell within the ability of those skilled in the art.

For any compound or inhibitory (e.g. antisense) nucleic acid describedherein, the therapeutically effective amount can be initially determinedfrom cell culture assays. Target concentrations will be thoseconcentrations of active compound(s) or inhibitory (e.g. antisense)nucleic acid(s) that are capable of achieving the methods describedherein, as measured using the methods described herein or known in theart.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring compounds or inhibitory (e.g. antisense) nucleic acidseffectiveness and adjusting the dosage upwards or downwards, asdescribed above. Adjusting the dose to achieve maximal efficacy inhumans based on the methods described above and other methods is wellwithin the capabilities of the ordinarily skilled artisan.

Dosages may be varied depending upon the requirements of the patient andthe compound or inhibitory (e.g. antisense) nucleic acid being employed.The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects. Determination of the proper dosage for a particularsituation is within the skill of the practitioner. Generally, treatmentis initiated with smaller dosages which are less than the optimum doseof the compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under circumstances is reached. In oneembodiment, the dosage range is 0.001% to 10% w/v. In anotherembodiment, the dosage range is 0.1% to 5% w/v.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound or inhibitory (e.g. antisense)nucleic acid effective for the particular clinical indication beingtreated. This will provide a therapeutic regimen that is commensuratewith the severity of the individual's disease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound or inhibitory (e.g. antisense) nucleicacid by considering factors such as compound or inhibitory (e.g.antisense) nucleic acid potency, relative bioavailability, patient bodyweight, presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

The ratio between toxicity and therapeutic effect for a particularcompound or inhibitory (e.g. antisense) nucleic acid is its therapeuticindex and can be expressed as the ratio between LD₅₀ (the amount ofcompound lethal in 50% of the population) and ED₅₀ (the amount ofcompound or inhibitory (e.g. antisense) nucleic acid effective in 50% ofthe population). Compounds or inhibitory (e.g. antisense) nucleic acidsthat exhibit high therapeutic indices are preferred. Therapeutic indexdata obtained from cell culture assays and/or animal studies can be usedin formulating a range of dosages for use in humans. The dosage of suchcompounds preferably lies within a range of plasma concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. See, e.g. Fingl et al., In: T HEPHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition and theparticular method in which the compound or inhibitory (e.g. antisense)nucleic acid is used.

Administration

The compositions of the present invention can be delivered bytransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols. For therapeutic applications,the compounds or inhibitory (e.g. antisense) nucleic acids or drugs ofthe present invention can be administered alone or co-administered incombination with conventional chemotherapy, radiotherapy, hormonaltherapy, and/or immunotherapy.

The compositions of the present invention can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe administered via intradermal injection of drug-containingmicrospheres, which slowly release subcutaneously (see Rao, J. BiomaterSci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gelformulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, asmicrospheres for oral administration (see, e.g., Eyles, J. Pharm.Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routesafford constant delivery for weeks or months.

The pharmaceutical compositions of the present invention can be providedas a salt and can be formed with many acids, including but not limitedto hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,etc. Pharmaceutical compositions described herein may be salts of acompound or inhibitory (e.g. antisense) nucleic acids or compositionwhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds or inhibitory (e.g.antisense) nucleic acids described herein. When compounds or inhibitory(e.g. antisense) nucleic acids of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds or inhibitory (e.g.antisense) nucleic acids with a sufficient amount of the desired base,either neat or in a suitable inert solvent. Examples of pharmaceuticallyacceptable base addition salts include sodium, potassium, calcium,ammonium, organic amino, or magnesium salt, or a similar salt. Whencompounds or inhibitory (e.g. antisense) nucleic acids of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19(1977)). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts. Otherpharmaceutically acceptable carriers known to those of skill in the artare suitable for the present invention. Salts tend to be more soluble inaqueous or other protonic solvents that are the corresponding free baseforms. In other cases, the preparation may be a lyophilized powder in 1mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

The neutral forms of the compounds or inhibitory (e.g. antisense)nucleic acids may be regenerated by contacting the salt with a base oracid and isolating the parent compound or inhibitory (e.g. antisense)nucleic acids in the conventional manner. The parent form of thecompound or inhibitory (e.g. antisense) nucleic acids differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

Certain compositions (compounds or inhibitory (e.g. antisense) nucleicacids described herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia),(IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic),(IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie),(IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments)) can exist in unsolvated forms as well as solvated forms,including hydrated forms. In general, the solvated forms are equivalentto unsolvated forms and are intended to be encompassed within the scopeof the present invention. Certain compounds or inhibitory (e.g.antisense) nucleic acids described herein (e.g. Formula (I), (II),(III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb),(IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId),(IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh),including embodiments) may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

In another embodiment, the compositions of the present invention areuseful for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly comprise asolution of the compositions of the present invention dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of the compositions ofthe present invention in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight, andthe like, in accordance with the particular mode of administrationselected and the patient's needs. For IV administration, the formulationcan be a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the formulations of the compositions of thepresent invention can be delivered by the use of liposomes which fusewith the cellular membrane or are endocytosed, i.e., by employingreceptor ligands attached to the liposome, that bind to surface membraneprotein receptors of the cell resulting in endocytosis. By usingliposomes, particularly where the liposome surface carries receptorligands specific for target cells, or are otherwise preferentiallydirected to a specific organ, one can focus the delivery of thecompositions of the present invention into the target cells in vivo.(See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn,Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989).

The compounds or inhibitory (e.g. antisense) nucleic acids describedherein can be used in combination with one another, with other activeagents known to be useful in treating a disease (e.g. cancer) associatedwith cells expressing a particular kinase (e.g. eEF-2K) or receptor(e.g. 5-HTR_(1B), 5-HTR_(1D), 5-HTR), or with adjunctive agents that maynot be effective alone, but may contribute to the efficacy of the activeagent.

In some embodiments, co-administration includes administering one activeagent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a secondactive agent. Co-administration includes administering two active agentssimultaneously, approximately simultaneously (e.g., within about 1, 5,10, 15, 20, or 30 minutes of each other), or sequentially in any order.In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding both active agents. In other embodiments, the active agentscan be formulated separately. In another embodiment, the active and/oradjunctive agents may be linked or conjugated to one another.

As a non-limiting example, the compounds or inhibitory (e.g. antisense)nucleic acids described herein (e.g. Formula (I), (II), (III), (IV),(V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments, or any compounds described in the Examples section) can beco-administered with conventional chemotherapeutic agents includingalkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil,busulfan, melphalan, mechlorethamine, uramustine, thiotepa,nitrosoureas, etc.), anti-metabolites (e.g., 5-fluorouracil,azathioprine, methotrexate, leucovorin, capecitabine, cytarabine,floxuridine, fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.),plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine,podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors(e.g., irinotecan, topotecan, amsacrine, etoposide (VP16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g.cisplatin, oxaloplatin, carboplatin, etc.), other kinase inhibitors,other 5-HTR modulators, and the like.

The compounds or inhibitory (e.g. antisense) nucleic acids describedherein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa),(IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc),(IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If),(IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments, or anycompounds described in the Examples section) can also be co-administeredwith conventional hormonal therapeutic agents including, but not limitedto, steroids (e.g., dexamethasone), finasteride, aromatase inhibitors,tamoxifen, and gonadotropin-releasing hormone agonists (GnRH) such asgoserelin.

Additionally, the compounds or inhibitory (e.g. antisense) nucleic acidsdescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe),(If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments, or anycompounds described in the Examples section) can be co-administered withconventional immunotherapeutic agents including, but not limited to,immunostimulants (e.g., Bacillus Calmette-Guérin (BCG), levamisole,interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g.,anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonalantibodies), immunotoxins (e.g., anti-CD33 monoclonalantibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.).

In a further embodiment, the compounds or inhibitory (e.g. antisense)nucleic acids described herein (e.g. Formula (I), (II), (III), (IV),(V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments, or any compounds described in the Examples section) can beco-administered with conventional radiotherapeutic agents including, butnot limited to radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y,⁹⁹Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques or maybe produced under sterile conditions. Aqueous solutions can be packagedfor use or filtered under aseptic conditions and lyophilized, thelyophilized preparation being combined with a sterile aqueous solutionprior to administration. The compositions can contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents, and the like, e.g., sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate, and triethanolamine oleate.

Formulations suitable for oral administration can comprise: (a) liquidsolutions, such as an effective amount of a packaged compound orinhibitory (e.g. antisense) nucleic acid described herein (e.g. Formula(I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib),(IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id),(IId), (IIId), (IVd), (Vd), (Ie), (Hie), (If), (IIIf), (Ig), (IIIg),(Ih), (IIIh), including embodiments, or any compounds described in theExamples section) or drug suspended in diluents, e.g., water, saline, orPEG 400; (b) capsules, sachets, or tablets, each containing apredetermined amount of a compound or inhibitory (e.g. antisense)nucleic acid described herein (e.g. Formula (I), (II), (III), (IV), (V),(Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb),(Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd),(Ie), (Hie), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), includingembodiments, or any compounds described in the Examples section), asliquids, solids, granules or gelatin; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, sucrose, mannitol, sorbitol, calcium phosphates, cornstarch, potato starch, microcrystalline cellulose, gelatin, colloidalsilicon dioxide, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise a compound or inhibitory (e.g. antisense) nucleic aciddescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe),(If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments, or anycompounds described in the Examples section) or drug in a flavor, e.g.,sucrose, as well as pastilles comprising the compounds or inhibitory(e.g. antisense) nucleic acids described herein (e.g. Formula (I), (II),(III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb),(IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId),(IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih), (IIIh),including embodiments, or any compounds described in the Examplessection) in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like, containing, in addition to thecompounds or inhibitory (e.g. antisense) nucleic acids described herein(e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa),(Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc),(Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf),(Ig), (IIIg), (Ih), (IIIh), including embodiments, or any compoundsdescribed in the Examples section), carriers known in the art.

The compounds or inhibitory (e.g. antisense) nucleic acids describedherein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa),(IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc),(IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If),(IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments, or anycompounds described in the Examples section) of choice, alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which comprises an effective amount of a packagedcompound or inhibitory (e.g. antisense) nucleic acid described herein(e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa),(Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc),(Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf),(Ig), (IIIg), (Ih), (IIIh), including embodiments, or any compoundsdescribed in the Examples section) or drug with a suppository base.Suitable suppository bases include natural or synthetic triglycerides orparaffin hydrocarbons. In addition, it is also possible to use gelatinrectal capsules which contain a combination of the compounds orinhibitory (e.g. antisense) nucleic acids described herein (e.g. Formula(I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib),(IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id),(IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg),(Ih), (IIIh), including embodiments, or any compounds described in theExamples section) or drug of choice with a base, including, for example,liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Injection solutions and suspensions can also beprepared from sterile powders, granules, and tablets. In the practice ofthe present invention, compositions can be administered, for example, byintravenous infusion, orally, topically, intraperitoneally,intravesically, or intrathecally. Parenteral administration, oraladministration, and intravenous administration are the preferred methodsof administration. The formulations of compounds or inhibitory (e.g.antisense) nucleic acids can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component, e.g., a compound orinhibitory (e.g. antisense) nucleic acid described herein (e.g. Formula(I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib),(IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id),(IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg),(Ih), (IIIh), including embodiments, or any compounds described in theExamples section). The unit dosage form can be a packaged preparation,the package containing discrete quantities of preparation, such aspacketed tablets, capsules, and powders in vials or ampoules. Also, theunit dosage form can be a capsule, tablet, cachet, or lozenge itself, orit can be the appropriate number of any of these in packaged form. Thecomposition can, if desired, also contain other compatible therapeuticagents.

In therapeutic use for the treatment of cancer or depression, compoundsor inhibitory (e.g. antisense) nucleic acids described herein (e.g.Formula (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va),(Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc),(Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig),(IIIg), (Ih), (IIIh), including embodiments, or any compounds describedin the Examples section) utilized in the pharmaceutical compositions ofthe present invention are administered at the initial dosage of about0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, orabout 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg,can be used. The dosages, however, may be varied depending upon therequirements of the patient, the severity of the condition beingtreated, and the compound or inhibitory (e.g. antisense) nucleic aciddescribed herein (e.g. Formula (I), (II), (III), (IV), (V), (Ia), (IIa),(IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb), (Vb), (Ic), (IIc),(IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd), (Vd), (Ie), (IIIe),(If), (IIIf), (Ig), (IIIg), (Ih), (IIIh), including embodiments, or anycompounds described in the Examples section) or drug being employed. Forexample, dosages can be empirically determined considering the type andstage of cancer diagnosed in a particular patient. The dose administeredto a patient, in the context of the present invention, should besufficient to affect a beneficial therapeutic response in the patientover time. The size of the dose will also be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a kinase or receptor modulator compound orinhibitory (e.g. antisense) nucleic acid in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound orinhibitory (e.g. antisense) nucleic acid described herein (e.g. Formula(I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib),(IIb), (IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id),(IId), (IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg),(Ih), (IIIh), including embodiments, or any compounds described in theExamples section). Thereafter, the dosage is increased by smallincrements until the optimum effect under circumstances is reached. Forconvenience, the total daily dosage may be divided and administered inportions during the day, if desired.

The compounds or inhibitory (e.g. antisense) nucleic acids describedherein can be used in combination with one another, with other activeagents known to be useful in treating cancer or depression or a diseasecapable of being treated with a compound or inhibitory (e.g. antisense)nucleic acid as described herein, with adjunctive agents that may not beeffective alone, but may contribute to the efficacy of the active agent(e.g. compounds or inhibitory (e.g. antisense) nucleic acids asdescribed herein).

In some embodiments of the compounds (e.g. Formula (I), (II), (III),(IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb), (IIIb), (IVb),(Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId), (IIId), (IVd),(Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih) or (IIIh)),pharmaceutical compositions, and/or methods described herein, thecompound is selected from any of the compounds described in the Examplessection herein. In some embodiments of the compounds (e.g. Formula (I),(II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), (Va), (Ib), (IIb),(IIIb), (IVb), (Vb), (Ic), (IIc), (IIIc), (IVc), (Vc), (Id), (IId),(IIId), (IVd), (Vd), (Ie), (IIIe), (If), (IIIf), (Ig), (IIIg), (Ih) or(IIIh)), pharmaceutical compositions, and/or methods described herein,the compound is selected from the group consisting of:

ADDITIONAL EMBODIMENTS

1. A compound having the formula:

wherein R^(1A) is independently hydrogen, halogen, —CX^(1A) ₃,—C(O)R^(7A), —C(O)—OR^(7A), —C(O)NR^(7A)R^(8A), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1B) is independently hydrogen, halogen,—CX^(1B) ₃, —C(O)R^(7B), —C(O)—OR^(7B), —C(O)NR^(7B)R^(8B), substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(2A) is independently hydrogen, halogen,—CX^(2A) ₃, —C(O)R^(9A), —C(O)—OR^(9A), —C(O)NR^(9A)R^(19A), substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(2B) is independently hydrogen, halogen,—CX^(2B) ₃, —C(O)R^(9B), —C(O)—OR^(9B), —C(O)NR^(9B)R^(10B,) substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; wherein R^(1A) and R^(2A) are optionallyjoined to form a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl; wherein R^(1B) and R^(2B) areoptionally joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R³ isindependently hydrogen, halogen, —CX³ ₃, —CN, —SO₂Cl, —SO₁R¹⁴,—SO_(k)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m), —NR¹¹R¹², —C(O)¹³, —C(O)—OR¹³, —O—C(O)—R¹³, —C(O)NR¹¹R¹²,—NR¹¹C(O)R¹³, —OR¹⁴, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R^(7A),R^(7B), R^(8A), R^(8B), R^(9A), R^(9B), R^(10A), R^(10B), R¹¹, R¹², R¹³,and R¹⁴ are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; A¹and A² are independently ═N— or ═CR³—; L is independently a bond, —O—,—NH—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, substituted or unsubstitutedheteroarylene.

or —O—(CH₂)_(p)—O—; k and m are independently 1 or 2; n is independentlyan integer from 0 to 4; p, v, and w are independently an integer from 1to 20; z1 and z2 are independently an integer from 0 to 3; X^(1A),X^(1B), X^(2A), X^(2B), and X³ are independently —Cl, —Br, —I, or —F.

2. A compound of embodiment 1 having the formula:

3. A compound of embodiment 1 having the formula:

4. A compound of embodiment 1 having the formula:

5. A compound of embodiment 1 having the formula:

6. A compound of embodiment 1 having the formula:

7. The compound of any one of embodiments 1 to 6, wherein R^(1A) isindependently —C(O)R^(7A) or —C(O)—OR^(7A); R^(1B) is independently—C(O)R^(7B) or —C(O)—OR^(7B); R^(7A) is independently hydrogen,substituted or unsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted2 to 20 membered heteroalkyl, substituted or unsubstituted C₃-C₈cycloalkyl, substituted or unsubstituted 3 to 8 memberedheterocycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, orsubstituted or unsubstituted 5 to 10 membered heteroaryl; and R^(7B) isindependently hydrogen, substituted or unsubstituted C₁-C₂₀ alkyl,substituted or unsubstituted 2 to 20 membered heteroalkyl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 3 to 8membered heterocycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, orsubstituted or unsubstituted 5 to 10 membered heteroaryl.

8. The compound of any one of embodiments 1 to 7, wherein R^(7A) isindependently hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl orsubstituted or unsubstituted 2 to 10 membered heteroalkyl.

9. The compound of any one of embodiments 1 to 8, wherein R^(7B) isindependently hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl orsubstituted or unsubstituted 2 to 10 membered heteroalkyl.

10. The compound of any one of embodiments 1 to 9, wherein R^(7A) isindependently hydrogen or substituted or unsubstituted C₁-C₁₀ alkyl.

11. The compound of any one of embodiments 1 to 10, wherein R^(7B) isindependently hydrogen or substituted or unsubstituted C₁-C₁₀ alkyl.

12. The compound of any one of embodiments 1 to 11, wherein R^(7A) isindependently methyl.

13. The compound of any one of embodiments 1 to 12, wherein R^(7B) isindependently methyl.

14. The compound of any one of embodiments 1 to 11, wherein R^(7A) isindependently hydrogen.

15. The compound of any one of embodiments 1 to 12, wherein R^(7B) isindependently hydrogen.

16. The compound of any one of embodiments 1 to 15, wherein R^(1A) is—C(O)R^(7A).

17. The compound of any one of embodiments 1 to 15, wherein R^(1B) is—C(O)R^(7B).

18. The compound of any one of embodiments 1 to 15, wherein R^(1A) is—C(O)—OR^(7A).

19. The compound of any one of embodiments 1 to 15, wherein R^(1B) is—C(O)—OR^(7B).

20. The compound of any one of embodiments 1 to 15, wherein R^(1A) isunsubstituted alkyl.

21. The compound of any one of embodiments 1 to 15, wherein R^(1B) isunsubstituted alkyl.

22. The compound of any one of embodiments 1 to 21, wherein R^(2A) isindependently —C(O)R^(9A) or —C(O)—OR^(9A); R^(2B) is independently—C(O)R^(9B) or —C(O)—OR^(9B); R^(9A) is independently hydrogen,substituted or unsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted2 to 20 membered heteroalkyl, substituted or unsubstituted C₃-C₈cycloalkyl, substituted or unsubstituted 3 to 8 memberedheterocycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, orsubstituted or unsubstituted 5 to 10 membered heteroaryl; and R^(9B) isindependently hydrogen, substituted or unsubstituted C₁-C₂₀ alkyl,substituted or unsubstituted 2 to 20 membered heteroalkyl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 3 to 8membered heterocycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, orsubstituted or unsubstituted 5 to 10 membered heteroaryl.

23. The compound of any one of embodiments 1 to 22, wherein R^(9A) isindependently hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl orsubstituted or unsubstituted 2 to 10 membered heteroalkyl.

24. The compound of any one of embodiments 1 to 23, wherein R^(9B) isindependently hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl orsubstituted or unsubstituted 2 to 10 membered heteroalkyl.

25. The compound of any one of embodiments 1 to 24, wherein R^(9A) isindependently hydrogen or substituted or unsubstituted C₁-C₁₀ alkyl.

26. The compound of any one of embodiments 1 to 25, wherein R^(9B) isindependently hydrogen or substituted or unsubstituted C₁-C₁₀ alkyl.

27. The compound of any one of embodiments 1 to 26, wherein R^(9A) ismethyl.

28. The compound of any one of embodiments 1 to 26, wherein R^(9B) ismethyl.

29. The compound of any one of embodiments 1 to 26, wherein R^(9A) ishydrogen.

30. The compound of any one of embodiments 1 to 26, wherein R^(9B) ishydrogen.

31. The compound of any one of embodiments 1 to 30, wherein R^(2A) is—C(O)R^(9A).

32. The compound of any one of embodiments 1 to 30, wherein R^(2B) is—C(O)R^(9B).

33. The compound of any one of embodiments 1 to 30, wherein R^(2A) is—C(O)—OR^(9A).

34. The compound of any one of embodiments 1 to 30, wherein R^(2B) is—C(O)—OR^(9B).

35. The compound of any one of embodiments 1 to 30, wherein R^(2A) isunsubstituted alkyl.

36. The compound of any one of embodiments 1 to 30, wherein R^(2B) isunsubstituted alkyl.

37. The compound of any one of embodiments 1 to 36, wherein R^(1A) andR^(2A) are joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl.

38. The compound of any one of embodiments 1 to 37, wherein R^(1B) andR^(2B) are joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl.

39. The compound of any one of embodiments 1 to 38, wherein R^(1A) andR^(2A) are joined to form a substituted or unsubstitutedheterocycloalkyl.

40. The compound of any one of embodiments 1 to 39, wherein R^(1B) andR^(2B) are joined to form a substituted or unsubstitutedheterocycloalkyl.

41. The compound of any one of embodiments 1 to 40, wherein R^(1A) andR^(2A) are joined to form a heterocycloalkyl fused to an aryl.

42. The compound of any one of embodiments 1 to 41, wherein R^(1B) andR^(2B) are joined to form a heterocycloalkyl fused to an aryl.

43. The compound of any one of embodiments 1 to 42, wherein R^(1A) andR^(2A) are joined to form a substituted or unsubstitutedisoindolin-2-yl-1,3-dione.

44. The compound of any one of embodiments 1 to 43, wherein R^(1B) andR^(2B) are joined to form a substituted or unsubstitutedisoindolin-2-yl-1,3-dione.

45. The compound of any one of embodiments 1 to 44, wherein R³ isindependently hydrogen, halogen, —C(O)R¹³, —O—C(O)—R¹³, —C(O)—OR¹³,—OR¹⁴, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

46. The compound of any one of embodiments 1 to 44, wherein R³ isindependently hydrogen —C(O)R¹³, —O—C(O)—R¹³, —C(O)—OR¹³, or —OR¹⁴; andR¹³ and R¹⁴ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

47. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl.

48. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently hydrogen.

49. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted or unsubstituted alkyl.

50. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted or unsubstituted C₁-C₂₀ alkyl.

51. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted or unsubstituted C₆-C₁₆ alkyl.

52. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently unsubstituted C₆-C₁₆ alkyl.

53. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted C₆-C₁₆ alkyl.

54. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently unsubstituted C₁₂-C₁₆ alkyl.

55. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted C₁₂-C₁₆ alkyl.

56. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted or unsubstituted heteroalkyl.

57. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted or unsubstituted 2 to 20 memberedheteroalkyl.

58. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted or unsubstituted 6 to 16 memberedheteroalkyl.

59. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently substituted 6 to 16 membered heteroalkyl.

60. The compound of any one of embodiments 1 to 46, wherein R¹³ and R¹⁴are independently unsubstituted 6 to 16 membered heteroalkyl.

61. The compound of any one of embodiments 1 to 46, wherein R¹⁴ is:

R⁴ is independently hydrogen, halogen, —CX⁴ ₃, —CN, —SO₂Cl, —SO_(q)R¹⁸,—SO_(r)NR¹⁵R¹⁶, —NHNH₂, —ONR¹⁵R¹⁶, —NHC═(O)NHNH₂, —NHC═(O)NR¹⁵R¹⁶,—N(O)_(t), —NR¹⁵R¹⁶, —C(O)R¹⁷, —C(O)—OR¹⁷, —C(O)NR¹⁵R¹⁶, —OR¹⁸,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R¹⁵, R¹⁶, R¹⁷, and R¹⁸ areindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; r and tare independently 1 or 2; q is independently an integer from 0 to 4; uis independently an integer from 0 to 5; and X⁴ is independently —Cl,—Br, —I, or —F.

62. The compound of any one of embodiments 1 to 45, wherein R³ isindependently halogen.

63 The compound of any one of embodiments 1 to 46, wherein R³ isindependently hydrogen.

64. The compound of any one of embodiments 1 to 63, wherein R³ isindependently —C(O)R¹³.

65. The compound of any one of embodiments 1 to 63, wherein R³ isindependently —O—C(O)—R¹³.

66. The compound of any one of embodiments 1 to 63, wherein R³ isindependently —C(O)—OR¹³.

67. The compound of any one of embodiments 1 to 63, wherein R³ isindependently —OR¹⁴.

68. The compound of any one of embodiments 1 to 46, wherein R³ isindependently —C(O)R¹³ or —O—C(O)—R¹³; R¹³ is

R⁴ is independently hydrogen, halogen, —CX⁴ ₃, —CN, —SO₂Cl, —SO_(q)R¹⁸,—SO_(r)NR¹⁵R¹⁶, —NHNH₂, —ONR¹⁵R¹⁶, —NHC═(O)NHNH₂, —NHC═(O)NR¹⁵R¹⁶,—N(O)_(t), —NR¹⁵R¹⁶, —C(O)R¹⁷, —C(O)—OR¹⁷, —C(O)NR¹⁵R¹⁶, —OR¹⁸,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R¹⁵, R¹⁶, R¹⁷, and R¹⁸ areindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; r and tare independently 1 or 2; q is independently an integer from 0 to 4; uis independently an integer from 0 to 5; and X⁴ is independently —Cl,—Br, —I, or —F.

69. The compound of any one of embodiments 1 to 68, wherein A¹ is ═CR³—.

70. The compound of any one of embodiments 1 to 69, wherein A² is ═CR³—.

71. The compound of any one of embodiments 1 to 68, wherein A¹ is ═N—.

72. The compound of any one of embodiments 1 to 69, wherein A² is ═N—.

73. The compound of any one of embodiments 1 to 72, wherein the compoundis not:

74. The compound of any one of embodiments 1 to 73, wherein the compoundis:

75. A pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and a compound of any one of embodiments 1 to 74.

76. A pharmaceutical composition comprising a pharmaceuticallyacceptable excipient, a liposome, and a compound of any one ofembodiments 1 to 74.

77. The pharmaceutical composition of embodiment 76, wherein theliposome comprises dioleoyl-sn-glycero-3-phosphocholine.

78. The pharmaceutical composition of embodiment 76, wherein theliposome comprises dimyristoyl-phosphatidylcholine.

79. The pharmaceutical composition of embodiment 76, wherein saidcompound is in said liposome.

80. The pharmaceutical composition of embodiment 76, wherein saidliposome comprises a targeting moiety.

81. The pharmaceutical composition of embodiment 80, wherein saidtargeting moiety is folate.

82. A method of treating a disease in a patient in need of suchtreatment, said method comprising administering a therapeuticallyeffective amount of a compound of any one of embodiments 1 to 74.

83. A method of treating a disease in a patient in need of suchtreatment, said method comprising administering a therapeuticallyeffective amount of a pharmaceutical composition of any one ofembodiments 75 to 81.

84. The method of any one of embodiments 82 to 83, wherein said diseaseis cancer.

85. The method of any one of embodiments 82 to 84, wherein said diseaseis breast cancer, ovarian cancer, pancreatic cancer, liver cancer,glioblastoma, glioma, lung cancer, prostate cancer, leukemia, ormelanoma.

86. The method of any one of embodiments 82 to 85, wherein said diseaseis metastatic cancer.

87. The method of any one of embodiments 82 to 83, wherein said diseaseis a migraine headache.

88. The method of any one of embodiments 82 to 83, wherein said diseaseis depression.

89. The method of any one of embodiments 82 to 88, wherein said compoundor pharmaceutical composition is co-administered with a chemotherapeuticagent.

EXAMPLES Example 1 CaMK-III is Overexpressed in Breast Cancer Cells

CaMK-III protein expression was assessed by Western blot analysis in thenon-tumorigenic breast epithelium MCF10A cell line and compared to otherbreast cancer cell lines. These included estrogen receptor-negative,ER(−) cells including MDA-MB-231, MDA-MB-435, BT549, BT20, and SK—BR3(HER2 overexpressing); ER(+) cells including MCF-7, MDA-MB-361 (HER2overexpressing), and T47D) and metastatic (MCF10CA1a; CA1a) breastcancer cells (FIG. 13A). Overall, CaMK-III levels were increased in allbreast cancer cell lines.

Cell Lines, Culture Conditions, and Reagents.

The human breast cancer cell lines were obtained from ATCC. Cells werecultured at 37° C. in DMEM supplemented with 5% FBS in a humid incubatorwith 5% CO₂.

Cell Viability and Growth Assays.

Viable and dead cells were detected by the trypan blue exclusion assay,the viability and/or proliferation of cells a MTS assay was used [AkarU, et al. Autophagy 2008: 4:669-79].

Plasmids.

EGB2T-CaMK-III (aka eEF-2K)—cDNA from the bacterial expression vectorp32TCaMK-III, encoding the human Trx-His6-tagged CaMK-III/eEf2K (GenBankaccession number NM_(—)013302), was used as a template in a PCR reaction(Franken N A, et al. Nat Protoc 2006: 1:2315-9). To clone the humanCaMK-III cDNA, the desired sequence was amplified by PCR using aspecifically designed forward primer, 5′-TTT GGTACC ATG GCA GAC GAA GATCTC ATC-3′ (SEQ ID NO:1) (KpnI recognition site underlined) and reverseprimer, 5′-AAA TGCGGCCGC TTA CTC CTC CAT CTG GGC CCA-3′ (SEQ ID NO:2)(NotI recognition site underlined), and ligated into the EGB-2T vector(Lu K P, Endocr Rev 1993: 14:40-58).

Western Blot Analysis.

After treatment the cells were collected and Western blot was performedas previously described [Akar U, et al. Autophagy 2008: 4:669-79]. Themembranes were blocked with 5% dry milk and probed with primaryantibodies anti-CaMK-III (eEF-2K) monoclonal antibody, p-EF2 (Thr-56),EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, pIGF-1R (Tyr-1131),IGF-1Rβ(#3027), p-Src (Tyr-416), p-paxillin (Tyr-31), p-mTOR (Ser-2448)(Cell signaling); VEGF, HIF1α, p-Fak(Tyr-397) (BD Transfection); c-myc,Bcl-2, caspase 9 (cleaved), poly ADP-ribose polymerase (PARP) (SantaCruz, Calif.). Horseradish peroxidase-conjugated anti-rabbit oranti-mouse secondary antibody (Amersham Life Science, Cleveland, Ohio).Mouse anti-β-actin and donkey anti-mouse secondary antibodies (SigmaChemical, St. Louis, Mo.) were used to monitor β-actin expression toensure equal loading of proteins.

Example 2 Knockdown of CaMK-III Inhibits Cell Proliferation of BreastCancer Cells

After determining that CaMK-III expression is increased in breast cancercell lines, we examined possible effect of CaMK-III on breast cancercell growth in vitro. First, we designed siRNA sequences to targetCaMK-III and assessed its ability to down-regulate CaMK-III expressionin cells. As shown in FIG. 13B, 50 and 75 nM of siRNA targeting CaMK-IIIefficiency decreased CaMK-III protein expression about 90% or more at 48h time point in MDA-MB231 cells Inhibition of CaMK-III by siRNA resultedin reduction of phosphorylation (Thr56) of its downstream target eEF2and significant inhibition of cell proliferation in MDA-MB231 cells(p<0.05)(FIGS. 13B and C). Similar results were obtained using MCF-7cells.

To further demonstrate that CaMK-III is involved in cell proliferationwe transiently transfected MDA-MB231 cells with GST-conjugated CaMK-IIIusing an expression vector containing DNA encoding for wild typeCaMK-III (FIG. 13D). CaMK-III overexpression significantly increasedproliferation of MDA-MB-231 cells compared with those transfected withempty control vector (p<0.05) (FIG. 13E). These data suggest thatCaMK-III is involved in proliferation of breast cancer cells.

Example 3 Knockdown of CaMK-III Inhibits the In Vitro Growth Rate andColony Formation of Breast Cancer Cells

To study the effect of CaMK-III growth of ER(−) and ER(+) breast cancercells we performed a clonogenic assay, which is based on the ability ofa single cell to grow into a colony (Franken N A, et al. Nat Protoc2006: 1:2315-9), using MDA-MB-231 and MCF-7 cells, respectively. Cellswere transfected with either CaMK-III or control siRNA (50 nM) every 4days over a period of two weeks. The knockdown of CaMK-III significantlyreduced growth rates and number of colonies (>75%) of both MDA-MB-231(FIG. 14A) and MCF-7 cells (FIG. 14B) compared to control siRNAtreatment (p<0.05). These data indicate that CaMK-III makes significantcontributions to breast cancer cell growth in vitro.

Clonogenic Survival Assay.

This assay is an in vitro cell survival assay based on the ability of asingle cell to grow into a colony. Briefly, cells (500 cells/well) weretransfected with control siRNA or CaMK-III siRNA every week and grownfor 2 to 3 weeks. Cells were stained with crystal violet and colonieswere counted. Each experiment was repeated twice.

Example 4 Knockdown of CaMK-III is Inhibits c-Myc, Cyclin D1, NF-kB andInduces p27^(Kip1) Expression

To gain a mechanistic insight into how CaMK-III inhibition leads togrowth inhibition, we knocked down CaMK-III by siRNA and examinedexpression of proteins critical to growth, cell cell cycle and survival.We first examined if CaMK-III inhibition induced cell death. As shown inFIG. 14C CaMK-III knockdown resulted in induction of apoptosis asindicated by PARP cleavage. The c-myc oncogene is amplified oroverexpressed in the majority of human breast cancers and contributes tocell proliferation and is an indicator of risk of recurrence and poorprognosis in patients (Liao D J, Dickson R B. Endocr Relat Cancer 2000:7:143-64). As shown in FIG. 14D, CaMK-knockdown significantly reducedc-myc expression in MDA-MB-231 cells compared to control cells treatedwith control siRNA. The cyclin D1 proto-oncogene and p27^(Kip1)cyclin-dependent kinase inhibitor (CDK) are important regulators of G1to S phase progression. Cyclin D1 plays a role in malignanttransformation and progression and the overexpression of cyclin D1 andthe reduced expression of p27^(Kip1) are indicative of a poor prognosticfactor in breast cancer (Musgrove E A. Growth Factors 2006: 24:13-9;Abukhdeir A M, Park B H. Expert Rev Mol Med 2008;:10:e19). Western blotanalysis revealed that knockdown of CaMK-III markedly reduced theexpression of cyclin D1 and increased the expression of p27^(Kip1) inMDA-MB-231cells (FIG. 14D). When combined with doxorubicin, a commonlyused chemotherapeutic agent, CaMK-III inhibition led to a furtherreduction in cyclin D1 and further enhancement of p27^(Kip1) expression(FIG. 18). The tumor suppressor PDCD4, which previously we have shown toup-regulate p27^(Kip1) expression (Ozpolat B, et al. Mol Cancer Res2007: 5:95-108) was induced by the down-regulation of CaMK-III (FIG.18).

Furthermore, we examined NF-κB, which is known to promote survival andangiogenesis and prevents apoptosis (Shibata A, et al. Breast cancerresearch and treatment 2002: 73:237-43). The knockdown of CaMK-III alsoreduced the expression of a transcriptionaly active form of NF-κB(p-p65-ser-356) (FIG. 14E). These data indicate that knockdown CaMK-IIIinhibits expression of key cellular proteins contributing growth andsurvival of breast cancer cells.

Transfections with siRNA and Plasmid.

Cells were transfected with siRNA as previously described [Akar U, etal. Autophagy 2008: 4:669-79]. siRNA sequence targeting CaMK-III wasdesigned using siRNA-designing software (Qiagen): CAMK-III (EF2KsiRNA)siRNA-1 #1, 5′-GCCAACCAGUACUACCAAA-3′ (SEQ ID NO:3). A previouslypublished siRNA sequence was also used target CAMK-III: siRNA#2;5′-AAGCUCGAACCAGAAUGUC-3 (SEQ ID NO:4) [Li H M, et al. Proc Amer AssocCancer Res 48: 4917, 2007]. Control non-silencing siRNA sequence5′-AAUUCUCCGAACGUGUCACGU-3′ (SEQ ID NO:5) [Abukhdeir A M, Park B H.Expert Rev Mol Med 2008;:10:e19]. siRNA targeting c-Src was purchasedfrom Sigma Aldrich. 1 μg of siRNA, mixed with the transfection reagent(Hyperfect Transfection Reagent, Qiagen), and added to each well.Plasmid vector (1-3 μg) containing human GST-tagged CaMK-III wastransfected into cells using the Qiagen plasmid transfection reagentaccording to the manufacturer's protocol.

Example 5 Depletion of CaMK-III Inhibits Invasion of MDA-MB-231 Cells

The ability of tumor cells to invade is one of the hallmarks of themetastatic phenotype. To determine a role for CaMK-III in mediatingbreast cancer cell invasion, in vitro invasion assays were performedusing Matrigel-coated Boyden chambers. This assay mimics the in vivoinvasion process and measures the number of cancer cells passing througha basement membrane matrix (Matrigel) towards media containingchemo-attractants (Shaw L M. Methods Mol Biol 2005: 294:97-105). Whiledepletion of CaMK-II by siRNA significantly reduced (˜80%) invasion ofMDA-MB231 cells (p<0.05) (FIG. 14F), the overexpression of CaMK-IIIsignificantly increases invasion of MDA-MB-231 breast cancer cells(p<0.05) (FIG. 14G). This suggests that inhibition of CaMK-III canreduce the invasion and metastatic potential of breast cancer cells.

Matrigel Invasion Assay.

MDA-MB-231 cells were transfected with CaMK-III or control siRNA and 24h later cells (1×10⁵ cells per well) were seeded onto Matrigel-coatedTranswell filters (8-μm pore size) in Matrigel invasion chambers (BDBiosciences, San Jose, Calif.), and the number of cells that invaded thelower side of the membrane were determined at 72 h by counting cells ina minimum of four randomly selected areas.

Example 6 Knockdown of CaMK-III Inhibits Activity of IGF-1R and PI3K/AktThrough Inhibition of c-Src in Breast Cancer Cells

Because the above data clearly implicated CaMK-III in the breast cancercell proliferation, invasion and survival, we examined the possibleinvolvement of c-Src signaling upon CaMK-III depletion andoverexpression. The Src family of non-receptor protein tyrosine kinasesare known to play critical roles in adhesion, migration/invasion,proliferation and resistance to chemotherapy (Finn R S. Ann Oncol 2008:19:1379-86). The knock-down of CaMK-III or c-Src by siRNA led tosignificant reduction of phospho-c-Src (Tyr-416)(FIG. 15A), andphospho-Fak (Tyr-397)(FIG. 15B) levels in MDA-MB231 cells, indicatingthat CaMK-III contributes to the promotion of the invasion phenotypethough the activation of c-Src/Fak signaling. Similar effects ofCaMK-III inhibition were observed on c-Src/Fak activation in doxorubicinresistant MCF-7 (MCF-7/DoxR) breast cancer cells (FIG. 19A). CaMK-IIIknockdown also led to a reduction in the phosphorylation of paxillin onTyr-31 (a target for Fak) in these cells (FIG. 19B). The overexpressionof CaMK-III resulted in a significant increase in p-Src (Tyr416) andp-eEF2(Thr56), suggesting that CaMK-III activity was increased inCaMK-III transfected MDA-MB-231 cells (FIG. 15C). This data indicatedthat CaMK-III participates in the activity of Src.

The insulin-like growth factors receptor 1 (IGF-1R) is phosphorylatedand activated by c-Src (19). More importantly, because IGF-1R plays acentral role in the development of mammary tumors, cell growth,differentiation, survival, and invasion/metastasis by regulatingproteins including c-myc, cyclin D1 and NF-κB we sought to determine ifIGF-1R is inhibited by CaMK-III and Src depletion. As shown in FIG. 15D,downregulation of CaMK-III resulted in a reduction of phosphorylatedIGF-1R (Tyr1131). The overexpression of CaMK-III results in an increasedlevel of IGF-1R autophosphorylation on Tyr-1131 (FIG. 15E). This datashow that IGF-1R may serve as a potential mediator of a CaMK-III/c-Srcaxis.

Src and IGF-1R signaling are known to induce PI3K/Akt (PKB) pathway,which is frequently increased in breast cancer and associated with apoor prognosis (Sun M, et al. The American journal of pathology 2001:159:431-7). Because our data suggest that CaMK-III and c-Src regulateIGF-1R signaling, we then examined whether CaMK-III plays a role in theactivity of Akt (Alessi D R, et al. The EMBO journal. 1996; 15:6541-51).The down-regulation of either CaMK-III or c-Src resulted in thedown-regulation of Akt activation (p-Ser473) (FIG. 15F) and mTORactivity (reduced p-Ser2448) (FIG. 19C), suggesting that CaMK-IIImediates Akt and mTOR activity, which plays essential roles in cellularresponses to growth factors and stress.

Example 7 Therapeutic Targeting of CaMK-III by Systemically AdministeredLiposomal-siRNA Inhibits Orthotopic Tumor Growth in a Breast CancerModel

The studies described above suggest that the CaMK-III gene enhances anumber of processes associated with tumorigenesis. To further assess itstherapeutic potential the effect of down-regulating CaMK-III in anorthotopic model of MDA-MB-231 breast cancer was assessed in nude mice.To eliminate potential off target-related effects two different siRNAswere used to target CaMK-III siRNA#1 (FIG. 13B) and siRNA#2 [Gross J M,Yee D. Cancer Metastasis Rev 2003: 22:327-36]. Targeting of the CaMK-IIIgene was achieved by systemic (i.v.) administration of DOPC-liposomalCaMK-III siRNA (150 μg/kg or about 4 μg/mouse) twice a week for a periodof 4 weeks. Treatment with both L-CaMK-III siRNA#1 and siRNA#2 inhibitedin vivo tumor growth. L-siRNA targeting of CaMK-III results insignificant down-regulation of its expression in the tumors (FIG. 22A).These data show that CaMK-III knockdown has growth inhibitory effects onbreast cancer tumor growth in vivo and therefore it may be a potentialtherapeutic target (FIG. 16C).

Moreover, no toxic effects were observed in mice exposed to liposomalCaMK-III siRNA for four weeks compared with the control group. Miceappeared healthy and did not lose weight during the 4 weeks of treatmentand the mean weight of L-CaMK-III siRNA and L-control-siRNA groups was28.3±0.8 g and 28.4±0.4 g, respectively.

Liposomal siRNA Preparation.

For in vivo delivery siRNA was incorporated intodioleoyl-sn-glycero-3-phosphocholine (DOPC). DOPC and siRNA were mixedin the presence of excess tertiary butanol at a ratio of 1:10 (w/w)siRNA/DOPC [Shaw L M. Methods Mol Biol 2005: 294:97-105]. Before in vivoadministration, this preparation was hydrated with normal 0.9% saline in100 μL for i.v injection per mouse.

Orthotopic Xenograft Tumor Model of Breast Cancer.

Athymic female nu/nu mice (5 week old) were obtained from the Departmentof Experimental Radiation Oncology at M. D. Anderson Cancer Center. Allstudies were conducted according to an experimental protocol approved bythe M. D. Anderson Institutional Animal Care and Use Committee.MDA-MB-231 cells (1×10⁻⁶) were injected into the right middle mammaryfat pad of each mouse. Two weeks after, when tumor size reached about3-5 mm liposomal siRNA treatments were initiated. Each mouse received150 μg/kg (equivalent of ˜4 μg/mouse) non-silencing control siRNA orCaMK-III siRNA twice a week (i.v. injection from the tail vein in 100 μgsaline) for four weeks. After finishing the treatment mice wereeuthanized by CO₂. Tumor tissues were removed for Western blot,Immunohistochemitry and TUNEL analysis and weighted to measure tumorgrowth.

Example 8 In Vivo Targeting of CaMK-III Enhances the Efficacy ofChemotherapy in a Breast Cancer Model

The in vitro studies described above demonstrate that the inhibition ofCaMK-III enhances doxorubicin-induced apoptosis. Doxorubicin is one ofthe most commonly used chemotherapies in cancer patients. To testwhether CaMK-III inhibition sensitizes breast cancer tumors in vivo tochemotherapy, a group of mice were orthotopicaly injected withMDA-MB-231 cells. Two weeks after tumor inoculation, mice were givenDOPC-L-CaMK-III siRNA twice a week as described above and doxorubicinonce a week (i.p, 4 mg/kg) for 4 weeks (FIG. 23). The group receivingcombination therapy (L-CaMK-III siRNA+doxorubicin) had the smallesttumors (p<0.05) compared to liposomal control siRNA+doxorubicin orL-CaMK-III siRNA groups (FIG. 16B). These findings suggest that thesilencing of CaMK-III enhances the effect of chemotherapy against breastcancer.

Example 9 In Vivo Targeting of CaMK-III Induces Apoptosis and InhibitsProteins Associated with Apoptosis, Invasion and Angiogenesis in BreastTumors

To determine the mechanisms by which CaMK-III inhibition induces tumorinhibition, tumor samples from in vivo therapeutic experiments wereexamined for induction apoptosis and proteins that may be critical totumor growth and progression. Western blot analysis and TUNEL assay oftumors demonstrated marked induction of apoptosis as evidenced byactivation of caspase-9 by cleavage and positive staining (brown),respectively (FIGS. 16C, D and E). Inhibition of anti-apoptotic proteinBcl-2 expression was noted in the tumor samples (FIG. 16C). In line withthe in vitro studies the phosphorylation of c-Src and Fak werediminished (FIG. 20A) in tumors after L-CaMK-III treatment. In addition,CaMK-III knockdown in tumors increased VEGF and doxorubicin-mediatedinhibition of HIF1α (FIG. 20B). We also found that CaMK-III inhibitionenhances doxorubicin-induced inhibition of Bcl-2 expression in thetumors (FIG. 20B), consistent with the possibility that Bcl-2 mediatessome of the effects of CaMK-III targeted therapy (Akar U, et al.Autophagy 2008: 4:669-79; Ozpolat B, et al. Proceedings of the AmericanAssociation for Cancer Research-AACR 2010: Abstract #51084).

Example 10 Downregulation of CaMK-III Leads to Apoptosis and EnhancesChemotherapy-Induced Cell Death in Drug Resistant Breast Cancer Cells

In vitro and in vivo inhibition of CaMK-III resulted in induction ofapoptosis (FIGS. 14D and 16C). This suggested a possible mechanism bywhich CaMK-III inhibition leads to growth inhibition and link betweenapoptosis protection and CaMK-III expression. To investigate whetherinhibition of CaMK-III sensitizes drug (doxorubicin)-resistant MCF7/DoxRand MDA-MB-231 cells to chemotherapy, cells were treated with eitherCaMK-III (50 nM) or control siRNA (50 nM) for 24 hours and then withdoxorubicin (ED₅₀ dose) for 48 hours. The ED50 of doxorubicin wasdetermined from a dose-escalation study in a proliferation assayInhibition of CaMK-III significantly enhanced doxorubicin-inducedapoptosis as indicated by caspase-9 cleavage in MCF7/DoxR and MDA-MB-231cells compared to controls (FIGS. 17A and B). To determine the percentof cells undergoing apoptosis in response to doxorubicin alone and incombination with CaMK-III siRNA was detected by Annexin V staining andFACS analysis. The inhibition of CaMK-III dramatically increasedapoptosis induced by doxorubicin (from 5% to 55%) in these MDAMB-231cells (FIG. 21).

Furthermore, downregulation of CaMK-III also enhanced paclitaxel-inducedinhibition of growth (FIG. 22). These data suggested that in vitro andin vivo the inhibition of CaMK-III inhibition sensitize cells tochemotherapy and enhanced the effects of chemotherapy.

Analysis of Cell Death.

Apoptosis was assessed by an Annexin V assay, monitoring PARP andcaspase 9 cleavage by Western blot. To provide a comparative assay ofapoptosis by Annexin V labeling, tumor cells (1×10⁶) treated withCaMK-III siRNA or control siRNA for 24 to 96 hours were harvested andwashed with PBS. Cells were resuspended in binding buffer and stainedwith Annexin V and propidium iodide (PI) according to the manufacturer'sprotocol (BD Pharmingen Annexin V kit, San Diego, Calif.). Positivecells were detected and quantified by FACS analysis.

TdT-Mediated dUTP Nick End Labeling (TUNEL) Assay.

Apoptotic events after treatment with L-siRNA and/or chemotherapyregimens were determined by the TdT-mediated dUTP nick end-labeling(TUNEL) assay (Promega, Madison, Wis.) in tumor sections as describedpreviously according to the manufacturer's protocol [Finn R S. Ann Oncol2008: 19:1379-86].

Statistical Analysis.

The data were expressed as the means±SD of three or more independentexperiments and statistical analysis was performed using the two-tailedand paired Student's t-test. P values less than 0.05 were consideredstatistically significant and indicated by an asterisk.

Example 11 Analysis of Examples 1 Through 10

Here we provide the first evidence that in vivo therapeutic targeting ofCaMK-III expression inhibits growth of established tumors in anorthotopic xenograft model of a highly aggressive and metastatic breastcancer. In addition, our data suggest that targeting CaMK-III may beused as a co-therapy for breast cancer to enhance efficacy of commonlyused chemotherapies, such as doxorubicin and paclitaxel.

CaMK-III is a protein kinase whose activity has been shown to be absentin normal tissue adjacent to breast cancer, but significantly increasedin breast cancer specimens using kinase assays (Parmer T G, et al. Br JCancer 1999: 79:59-64). CaMK-III is selectively activated inproliferating cells and especially in the S-phase of the cell cycle(Parmer T G, et al. Cell Growth Differ 1997: 8:327-34). The datapresented here demonstrate that CaMK-III is involved in breast cancercell growth, invasion and sensitize cells to chemotherapy, suggestingthat it may be a useful novel therapeutic target in breast cancer.

Our data suggest that siRNA-mediated knockdown of CaMK-III inhibitscolony formation in both ER(+) and ER(−) breast cancer cells.Conversely, ectopic expression of CaMK-III significantly increasesproliferation of MDA-MB-231 cells in vitro; Our findings, for the firsttime, show that CaMK-III is involved in the expression of two importantregulators of the cell cycle, namely c-myc, cyclin D1 and p27^(Kip1);inhibiting the expression of the c-myc and cyclin D1, and concomitantlyinducing the expression of p27^(Kip1). C-myc and cyclin D1, whichpromotes entry of cells into S phase, induce proliferation, can causemalignant transformation, and is overexpressed in more than 50% ofbreast cancer patients (Gillett C, et al. Cancer Res 1994: 54:1812-7).p27^(Kip1) is a CDK-inhibitor and a key regulator of G1-to-S phaseprogression (Alkarain A, et al. J Mammary Gland Biol Neoplasia 2004:9:67-80).

c-Src is a non-receptor tyrosine kinase, which is over-activated in >70%of breast cancers and recently has been proposed as a target for breastcancer. c-Src plays a critical role in tumorigenesis including,invasion/metastasis, proliferation and angiogenesis as well aschemo-resistance (Finn R S. Ann Oncol 2008: 19:1379-86). Notably, itsactivity is associated with the worst outcomes in breast cancer patients(Wilson G R, et al. Br J Cancer 2006: 95:1410-4). Our findings suggestthat down-regulation of CaMK-III significantly inhibits-80% of invasionof breast cancer cells (FIG. 14F). The overexpression of CaMK-IIIsignificantly increases migration and invasion of MDA-MB-231 cells,suggesting that CaMK-III participates to this process. Knockdown ofCaMK-III significantly reduced tyrosine phosphorylation of Src/FAK andPaxillin activity in MDA-MB231 and MCF7/DoxR cells (Finn R S. Ann Oncol2008: 19:1379-86). Src and FAK are activated by RTKs and integins (FinnR S. Ann Oncol 2008: 19:1379-86). This seems to occur through anindirect mechanisms or a mediator since CaMK-III is a serine threoninekinase. CaMK-III has been suggested to inhibit translation transientlyby phosphorylation of eEF2 (Ryazanov A G, et al. FEBS Lett 1991:285:170-5). Transient inhibition of protein synthesis is an early eventfollowing mitogenic stimulation (Celis J E, et al., Proc Natl Acad SciUSA 1990: 87:4231-5) and has been shown to result in preferentialelimination of proteins with short-half lives (White S J, et al. FreeRadic Biol Med 2007: 43:1313-21; Tuynder M, et al., Proc Natl Acad SciUSA 2004: 101:15364-9).

Experiments examining the effect of CaMK-III inhibition andoverexpression suggest that CaMK-III constitutively induces theactivation of IGF-1R through c-Src-mediated phosphorylation of Tyr-1131,which is known to activate IGF-1R (Peterson J E, et al. The Journal ofbiological chemistry 1996: 271:31562-71). Despite its emerging role inbreast and other cancers, targeting IGF-1R has only recently undergonedevelopment as a molecular cancer therapeutic (Rosenzweig S A, Atreya HS. Biochem Pharmacol 2010: 80:1115-24). IGF-1R mediates bothpro-survival and anti-apoptotic signals and is known to play a role inresistance to chemo and radiation therapies (Rosenzweig S A, Atreya H S.Biochem Pharmacol 2010: 80:1115-24). Furthermore, IGF-1R activation isknown to cause transformation of breast epithelium and to increase thetumorigenic potential of a number of cancers (Ozpolat B, et al.Proceedings of the American Association for Cancer Research-AACR 2010:Abstract #51084). The present study suggests that the inhibition ofCaMK-III may significantly reduce IGF-1R activity through inhibition ofSrc (FIG. 15A), suggesting that targeting CaMK-III would have broadtherapeutic effects in patients with c-Src and IGF-1Rsignaling-dependent cancers. Both Src and IGF1R can activate thePI3K/Akt (PKB) pathway, which plays a pivotal role in cell survival andproliferation through a number of downstream effectors and promotesbreast cancer cell resistance to chemotherapy, trastuzumab, andtamoxifen (Castaneda C A, et al. Cancer metastasis reviews 2010:29:751-9). Our findings suggest that targeting CaMK-III inhibits Akt,potentially through c-Src, thereby contributing to the apoptotic effectsof inhibiting CaMK-III.

Furthermore, in vivo inhibition of CaMK-III reduced the expression ofBcl-2, which may participate in the induction of apoptosis in tumors(Akar U, et al. Autophagy 2008: 4:669-79; Oltersdorf T, et al., Nature2005 435:677-81). In fact, we have shown that in vivo targeted silencingof Bcl-2 by liposomal Bcl-2 siRNA inhibits growth of Bcl-2-expressingbreast cancer tumors, including MCF7 and MDA-MB-231 tumors in nude mice(Ozpolat B, et al. Proceedings of the American Association for CancerResearch-AACR 2010: Abstract #51084). Therefore in vivo inhibition ofBcl-2 in response to CaMK-III silencing may be important for tumorgrowth inhibition and enhanced response to chemotherapy.

Our data suggest that the in vivo inhibition of CaMK-III in tumorsdown-regulates the transcriptionally active form of NF-κB, p-p65(Ser-536) and VEGF expression and facilitates doxorubicin-induceddown-regulation of HIF1α, which are pivotal components of angiogenesisand have been validated as clinical targets in many tumors, includingbreast cancer. Increased expression of these molecules are linked to theactivation of c-Src in breast cells through NF-κB, which is induced byIGF-1R and regulates Bcl-2 (18). Since the down-regulation of NF-κBsignaling has been demonstrated to inhibit in vitro and in vivoexpression of VEGF, (23) it is possible that CaMK-III may mediate itsaffects on VEGF expression through the modulation of NF-κB in breastcancer cells.

Example 12 5-HT Induces Proliferation of Breast Cancer Cells

Serotonin (5-HT) has been shown to act as a growth factor on severalnon-tumoral cells (e.g. vascular smooth muscle cells, lung fibroblasts,renal mesangial cells) [Seuwen K, Pouyssegur J Biochem Pharmacol 1990:39: 985-990; Nemecek G M, et al. Stimulation of aortic smooth musclecell mitogenesis by serotonin. Proceedings of the National Academy ofSciences of the United States of America 1986: 83: 674-678; Takuwa N, etal. The American journal of physiology 1989: 257: F431-439] and induceproliferation in some cancer cell lines, including human lung carcinoma,hepatocellular carcinoma, pancreatic carcinoic cells [Siddiqui E J, etal. BJU Int 2006: 97: 634-639; Ishizuka J, et al., Journal of cellularphysiology 1992: 150: 1-7; Soll C, et al. Hepatology 2010: 51:1244-1254; Cattaneo M G, et al. European journal of pharmacology 1995:291: 209-211]. However, its role in breast cancer proliferation in notwell understood. To demonstrate that 5-HT plays a role in proliferationin breast cancer cells we treated the triple negative (ER−, PR− andHer2/neu−) highly metastatic and aggressive breast cancer cell lineMDA-MB-231 with serotonin (FIG. 24). Serotonin also inducedproliferation of ER+MCF-7 breast cancer cells.

Cell Lines, Culture Conditions, and Reagents.

The human breast cancer cell lines MDA-MB-231 and MCF-7/R (drugresistant) were obtained from ATTC and Dr. Kapil Mehta, PhD (MD AndersonCancer Center), respectively. Cells were cultured at 37° C. in DMEMsupplemented with 5% FBS in a humid incubator with 5% CO₂. For cellproliferation experiments, cells were seeded at a density of 1-2×10⁵cells in T-25 tissue culture flasks. Adherent cells were collected bytrypsinization, and cell numbers were determined using a Neubauer cellcounting chamber. All experiments were repeated at least three times.

Example 13 5-HT1B Receptor is Over Expressed in Breast Cancer Cell Lines

To elucidate the mechanism of 5-HT-induced cell proliferation in breastcancer cells, 5-HTR1B receptor protein expression was assessed byWestern blot analysis in a normal breast epithelium MCF10A cell line andcompared to other breast cancer cell lines. These included the estrogenreceptor-negative human ER(−) breast cancer cell lines, MDA-MB-231,MDA-MB-435 and the estrogen receptor-positive ER(+) breast cancer celllines, MCF-7, chemotherapy (doxorubicin) resistant MCF-7/Dox-R cells,MCF10CA1a (CA1a), a metastatic human breast cancer line. All breastcancer cell lines showed higher expression of 5-HTR1B than the normalbreast epithelium MCF10A cell line (FIG. 25), suggesting that 5-HT1Breceptor protein levels are upregulated in breast cancer cells.

In our studies we found that 5-HT1B receptor is over expressed in almostall Estrogen receptor ER(+), ER (−) highly aggressive and metastaticcells as well as chemotherapy (doxorubicin) resistant breast cancercells compared to normal breast epithelium cells (MCF10A). We also foundthat that serotonin induces proliferation of breast cancer cells andthat a specific inhibition of the serotonin 5-HT1B receptors by siRNAand a pharmacological inhibitor inhibits proliferation, colony formationand induce significant apoptosis and autophagic cell death.

Several 5-HT receptors (1D, 1F, 2C and 3A) were found to beover-expressed in some breast cancer cells, compared to MCF10A normalbreast epithelium (Pai V P, et al., Breast Cancer 2009: Res 11: R81). Inaddition to the 5-HT_(1D) receptor we found, for the first time that the5-HT_(1B) receptor is also over-expressed in ER(−), ER(+) and drugresistant breast cancer cells (FIG. 3E). Preliminary studies withSB-224289 a selective 5-HT_(1B) receptor antagonist (Selkirk et al.,1998) provided the first insight into a novel pathway for the regulationof the activity of several signaling pathways by the 5-HT1B serotoninreceptor. We found that SB224289 induces apoptosis and inhibits theSTAT3 and Bcl-2 pathways, as well as the PI3K/Akt pathway. These areactivated in the majority of breast cancers.

Western Blot Analysis.

After treatment the cells were trypsinized and collected bycentrifugation, and whole-cell lysates were obtained using a lysisbuffer as described previously [Akar U, et al. Mol Cancer Res 2007: 5:241-249]. Total protein concentration was determined using a DC proteinassay kit (Bio-Rad, Hercules, Calif.). Aliquots containing 30 g of totalprotein from each sample were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 4-20%gradient and electrotransferred to nitrocellulose membranes. Themembranes were blocked with 5% dry milk in Tris-buffered saline-Tween 20(TBST), probed with primary antibodies anti EREK1/2 or p-ERK1/2 (Cellsignaling), polyclonal LC3 antibody (Novus biologicals, Littleton,Colo.). The antibodies were diluted in TBST containing 2.5% dry milk andincubated at 4 C overnight. After the membranes were washed with TBST,they were incubated with horseradish peroxidase-conjugated anti-rabbitor anti-mouse secondary antibody (Amersham Life Science, Cleveland,Ohio). Mouse anti-actin and donkey anti-mouse secondary antibodies(Sigma Chemical, St. Louis, Mo.) were used to monitor-actin expressionto ensure equal loading of proteins. Chemiluminescent detection wasperformed with Chemi-glow detection reagents (Alpha Innotech, SanLeandro, Calif.). The blots were visualized with a FluorChem 8900 imagerand quantified by a densitometer using the Alpha Imager applicationprogram (Alpha Innotech, San Leandro, Calif.).

Example 14 Specific Inhibition of the 5-HT1B Receptor InhibitsProliferation of Breast Cancer Cells

To determine the role of the 5-HT1B receptor we inhibited 5-HTR1Bsignaling in breast cancer cells using a specific antagonist SB224289and assessed cell proliferation of [23]. This resulted in adose-dependent inhibition of MCF7, MB-231 and MCF7/Dox resistant cellproliferation as detected by an MTS assay (FIG. 26A-C). Another specificantagonist (SB216641) of 5-HTR1B also showed a similar ability toinhibit the growth of breast cancer cells.

Cell Viability and Growth Assays.

Viable and dead cells were detected by the trypan blue exclusion assay,where viable cells (non-stained) with intact membrane are able toexclude the dye, while dead cells take up the coloring blue agent fromthe media. To quantitatively assess the viability and/or proliferationof cells a MTS assay was used, which incorporates a redox indicator thatchanges color in response to metabolic activity. To study cell viabilityin response to CaMK-III siRNA or control siRNA and plasmid encodingCaMK-III or empty plasmid transfection cells were seeded in 96-wellplates at a density of 3×103 cells per well in 100 μl of medium. Nextday the medium was removed and cells were transfected with siRNA atdifferent concentrations in 100 μl of medium plus transfection mix. Twodays after transfection, the medium was removed and replaced by 180 μlof medium plus 20 μl of MTS and the cells were incubated under normalconditions until the color changed. Plates were read at dual wavelengths(570-595 nm) in an Elisa plate reader (Kinetic Microplate Reader,Molecular Devices Corporation, Sunnyvale, Calif.).

Example 15 Inhibition of 5-HT1B Leads to Reduce Colony Formation ofBreast Cancer Cells

To study the effect of 5-HT1B on the survival and colony formation ofbreast cancer cells a clonogenic survival assay was performed usingvarious concentrations of SB224259 over a period of two weeks. Thisassay is based on the ability of a single cell to grow into a colony[Franken N A, et al. Nat Protoc 2006: 1: 2315-2319]. The inhibition of5-HT1B significantly inhibited the formation of MCF7 cell colonies in adose dependent manner (FIG. 26D). Thus, compared to control experiments(untreated), SB224259-treated cells grew more slowly and produced fewercolonies. We found similar effects in MDA-MB-231 cells with SB224289.Overall, these data suggest that 5-HTR1B contributes to colony formationas inhibition of this receptor reduced colonies formed by breast cancercells. FIG. 27 shows the appearance of cells after 72 h of treatment ofMDA-MB231 cells. Cell death and detachment as well as reduced cellnumber was observed.

Clonogenic Survival Assay.

This assay is an in vitro cell survival assay based on the ability of asingle cell to grow into a colony. Briefly, cells (500cells/well) weretransfected with different siRNA as described above and at 48 hpost-transfection, cells were trypsinized and seeded into 6-well platesin the medium (500 cells/mL). Cells were transfected with control siRNAor CaMK-III siRNA every week and grown for 2 to 3 weeks. Cells werestained with crystal violet and colonies cells were counted. Each groupwas assayed in triplicate dishes, and each experiment was repeatedtwice.

Example 16 Inhibition of the 5-HT1B Receptor Induces Autophagy andApoptosis in Breast Cancer Cells

To determine the role of serotonin 5-HT1B receptor in cell survival andthe mechanism of growth inhibition induced by HT1B inhibitors weinvestigated cell death in breast cancer cells. When 5-HT1B wasinhibited by either SB224289 or SB216641 we detected marked induction ofautophagy (FIG. 28A) as detected by acridine orange staining, apoptosisby Annexin V and necrosis by propidium iodide (PI) staining and FACSanalysis (FIG. 28B) in MCF7 breast cancer cells. Induction of acidicvacuoles by acridine orange staining was also observed in MDA-MB231cells after SB224289 treatment for 72 hours (p<0.05) (FIG. 28C).Quantification of acridine orange stained vesicular organelles by flowcytometry indicated that the percentage of positive cells in SB224289 orSB216641-treated cells (23% and 17%, respectively) were significantlyhigher than in the control untreated cells (2%). SB224289 treatment alsoinduced apoptosis in a dose dependent manner in MCF cells (FIG. 28B). Asthe dose was increased SB224289 (higher than EC₅₀ 4.4 μM) a shift fromautophagy to apoptosis was observed, with reduced autophagy. Inductionof autophagy was further evidenced by induction of LC3-II, a specificmarker of autophagy in response to SB224284 in MCF7 and doxorubicinresistant MCF7 cells (MCF7/Dox-R) (FIG. 29A).

To further demonstrate that inhibition of HTR1B inhibition leads toautophagy in breast cancer cells, we transfected MCF-7 cells with greenfluorescent protein-tagged LC3 (GFP-LC3) expression plasmid anddetermined the accumulation of GFP-LC-II protein in autophagosomes aftertreatment with HTR1B inhibitor SB224289 (FIG. 29B). When autophagy isinduced, LC3-II, a cleaved product of LC3, specifically localizes to themembrane of autophagosomes [Akar U, et al. Autophagy 2008: 4: 669-679].Therefore accumulation of GFP-LC3-II in the vacuoles following theSB224289 treatment indicates formation of autophagosomes and inductionof autophagy in the cells (FIG. 29B a-b). In control or untreated cells,none of the above changes were observed except diffuse expressionpattern of GFP-LC3 detected by fluorescence microscopy. In HTR1Binhibitor treated cells, GFP-LC3 distributes from a diffuse cytoplasmicpattern to form punctuate structures that indicate preautophagosomal andautophagosomal membranes. FIG. 29Bc-d shows pictures of the cells withformation of autophagic vacuoles by phase contrast microscopy.

To further support the findings we analyzed induction of autophagy bytransmission electron microscopy, which clearly demonstrated thatinhibition of HT1B by SB224289 or SB216641 treatment induced formationof autophagic vesicles containing cellular organelles, with merging ofautophagic vesicles with lysosomes and lysed cellular content in theautophagosomes, indicating activity of lysosomal function anddegradation (FIG. 30). These results suggest that inhibition of HTR1Binduces autophagy breast cancer cells.

Analysis of Cell Death.

Apoptosis was assessed by an Annexin V assay, monitoring PARP andcaspase 9 cleavage by Western blot. To provide a comparative assay ofapoptosis by Annexin V labeling, tumor cells (1×10⁶) treated withCaMK-III siRNA or control siRNA for 24 to 96 hours were harvested andwashed with PBS. Cells were resuspended in binding buffer and stainedwith Annexin V and propidium iodide (PI) according to the manufacturer'sprotocol (BD Pharmingen Annexin V kit, San Diego, Calif.). Positivecells were detected and quantified by FACS analysis.

Evaluation of Autophagy by Detection of Acidic Vesicular Organelles.

To detect and quantify acidic vesicular organelles, we stained cellswith acridine orange as described previously.³¹ The number of acridineorange-positive cells was determined by fluorescence-activated cellsorting (FACS) analysis. Cell morphology was examined using fluorescencemicroscopy (Nikon, Melville, N.Y.) with the cells remaining in theirculture flasks.

Transmission Electron Microscopy.

MCF-7 cells were grown on six-well plates, treated with Bcl-2 siRNA orcontrol siRNA or left untreated, fixed for 2 hours with 2.5%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), postfixed in 1% OsO₄in the same buffer, and then subjected to the electron microscopicanalysis as described previously [Akar U, et al. Mol Cancer Res 2007: 5:241-249]. Representative areas were chosen for ultra-thin sectioning andviewed with a Hitachi 7600.

Statistical Analysis.

The data were expressed as the means±SD of three or more independentexperiments and statistical analysis was performed using the two-tailedand paired Student's t-test. P values less than 0.05 were consideredstatistically significant and indicated by an asterisk.

Example 17 5-HT1B Modulates Autophagy Through Activity of ERK1/2-MAPKSignaling in Breast Cancer Cells

Induction of ERK1/2 has been shown to participate in the induction ofautophagy. We investigated whether inhibition of HT1B induces ERK-MAPKsignaling in breast cancer cells. Blockage of the 5-HT1B receptor withSB224289 induced phosphorylation of ERK (p42/p44) in MCF7, MCF7/Doxresistant and MDA-MB-231 as detected by Western blot analysis (FIG.31-32). SB216641 also induced phosphorylation of ERK (p42/p44) andLC3-III in MCF7 cells (FIG. 32A). Induction of ERK signaling was closelyassociated with induction of autophagy marker LC3-II in all treatmentsand breast cancer cell lines tested including MDA-MB231 in response toinhibition of HT1B signaling by 2 different inhibitors.

To demonstrate a link between autophagy induction in response to HTR1Binhibition and ERK activation we inhibited ERK using a MEK inhibitor(PD98059) we transfected MCF-7 cells with green fluorescentprotein-tagged LC3 (GFP-LC3) expression plasmid and determined theaccumulation of GFP-LC-II protein in autophagosomes after treatment withHTR1B inhibitor SB224289 (FIG. 33A-B). Accumulation of GFP-LC3-II in thevacuoles following pretreatment with PD98059 prevented SB224289 inducedformation of autophagosomes and punctuate pattern indicating inductionof autophagy in the cells (FIG. 29B a-b) detected by fluorescencemicroscopy. In HTR1B inhibitor treated cells, GFP-LC3 showed punctuatestructures that indicate preautophagosomal and autophagosomal membranes.FIG. 33B shows number of GFP-LC3 positive cells formation of autophagicvacuoles with or without the treatment of ERK inhibitor.

To further demonstrate that autophagy induced in response to HTR1Binhibition is mediated by ERK activation we inhibited ERK using a MEKinhibitor (PD98059) and found that inhibition of ERK significantlyreduced autophagy induction in MCF7 and MDA-MB231 cells detected byacridine orange staining and FACS analysis (p<0.05) (FIGS. 34A and 34B).The percent positive cells with autophagy induced by SB224359 wasdecreased to 18% from 29.2% by ERK inhibitor, suggesting that ERKmediates participates to autophagy induced by inhibition of HTR1B.

Example 18 Induction of Autophagy by HT1B Inhibition Contributes to CellDeath

Autophagy has been shown to function as a protective survival pathway inresponse to nutrient or growth factor deprivation, hypoxia, metabolicand therapeutic stress [Dalby K N, et al. Autophagy 2010: 6: 322-329].It was also reported that autophagy induction can lead to cell death[Kanzawa T, et al. Cancer research 2003: 63: 2103-2108] [Akar U, et al.Mol Cancer Res 2007: 5: 241-249]. To understand the role of autophagy inresponse to HT1B inhibition by SB224289 we inhibited autophagy by 3-MA(FIG. 35A). The incidence of autophagy induced by SB224359 was decreasedto 12% from 22.4% by 3-MA. Cell viability was significantly increased by3-MA from 42% by SB224289 to 61% and 31% by SB216641 to 71% in MCF7cells (p<0.05) (FIG. 35B). These results suggest that inhibition ofautophagy increases cell viability by HTR1B inhibition and autophagycontributes to cell death.

Example 19 Analysis of Examples 12 Through 18

Serotonin (5-HT), a monoamine neurotransmitter, plays a mitogenic rolein some cancer cells. Serotonin mediates its effects through 14different receptors that are differentially expressed in differenttissues. In this study we demonstrated that serotonin inducesproliferation of breast cancer cells. Here we showed the expression androle of 5-HT1B receptor is markedly (about 8-10 fold) increased inbreast cancer cells. 5-HT1B receptor overexpression was found in ER(+),ER (−) highly aggressive and metastatic breast cancer cell linescompared to normal untranformed breast epithelium. More importantly, weshowed 5-HT1B mediates proliferation, survival, and autophagy in breastcancer cells and inhibition of the serotonin 5-HTR-1B by specificinhibitors significantly inhibited growth of ER(+), ER(−) and drug(doxorubicin) resistant MCF7/Dox-R cells inducing both authophagic andapoptotic cell death, which represent a novel mechanism of action byinhibitors and the role of these receptors in cell survival and death.

Inhibition of serotonin 5-HTR-1B also significantly inhibited colonyformation of MDA-MB-231 cells, which are considered triple negative(ER−, PR− and Her2−) and resistant to most therapies Inhibition of the5-HT1B also was effective in inhibition of proliferation of chemotherapy(doxorubicin) resistant celss (MCF7/DoxR), suggesting that targetingthis receptor may provide therapeutic benefit to drug resistant advancestage breast cancers. Specific targeting of HTR1B two differentinhibitors (SB224284 or SB216641) exerted similar effects and inducedsignificant apoptosis and autophagy, which was evidenced by electronmicroscopy, LC3-II expression, a hallmark of autophagy and GFP-LC3translocation to the autophagosomes. Inhibition of autophagy reducedcell death and increased cell survival in response to HTR1B inhibitors,suggesting that autophagy is involved in cell death induced by HTR1Binhibition in breast cancer cells. Induction of autophagy was associatedinduction of MAPK (ERK1/2) activity (FIG. 31 A, B, FIG. 32A). We foundERK activation participates to autophagy induction in response to HTR1Breceptor inhibition. ERK activation has been shown to induce autophagyin some cancer cells.

Current literature suggest that autophagic cell death can helpelimination of cancer cells following various treatment [Tsujimoto Y,Shimizu S Cell death and differentiation 2005:12 Suppl 2: 1528-1534][Kanzawa T, et al. Cancer research 2003: 63: 2103-2108] [Akar U, et al.Autophagy 2008: 4: 669-679]. Depending on the stimulus autophagy maycontribute to cell death or be associated with cell death [Kroemer G,Levine B Nature reviews Molecular cell biology 2008: 9: 1004-1010]. Ourstudy suggest that in breast cancer cells inhibition of HTR1B growthsignaling induces autophagy which participates in cell death.

In conclusion, our findings suggest for the first time that thatserotonin 5-HT1B receptors are overexpressed in breast cancer cells andHTR-1B signaling promotes growth and survival of breast cancer cells andthat inhibition of these receptors inhibits cell proliferation andinduces apoptosis and autophagic cell death.

Example 20 Triptans and Derivatives as Cancer Therapeutics

Our studies show that the serotonin 5-HT_(1B) receptor is over-expressedin primary, metastatic and resistant breast cancer cells and that invitro modulation of 5-HT_(1B) signaling in breast cancer cells inhibitsproliferation and induces apoptosis. Remarkably, a clinically safetriptan, 5-NT, a potent agonist of the 5-HT_(1B/1D) receptors exhibitsefficacy in both in vitro and in vivo breast cancer models.

Our studies in MCF-7 cells have established that 5-NT down-regulatesERK/cyclin-D, Akt/mTor, IGF1-R and Src/Fak and eEF-2K within 4 hours(FIG. 9). These represent some of the most important therapeutic targetsin breast cancer and may underlie the anti-proliferative andpro-apoptotic effects of 5-NT (FIG. 6). Recently, we discovered that thedown-regulation of eEF-2K (which is over-expressed in all tumorigenicbreast cancer lines tested) disrupts these signaling pathways in both invitro and in vivo breast cancer models, suggesting that eEF-2K may be animportant target for 5-NT therapy. Ovarian cancer where our studiessuggests that the 5-HT1B receptor is also over-expressed.

Synthetic procedures for the elaboration of the 5-hydroxytryptaminescaffold are well-established (Glennon, 1996; Glennon et al., 1996;Glennon et al., 1994; Halazy et al., 1996; John et al., 1999; Perez etal., 1995; Perez et al., 1998a; Perez et al., 1998b). In addition,because 5-HT_(1B/1D) receptors form heterodimers dimers (Xie et al.,1999), dimeric derivatives of 5-NT which have been shown to potentlybind 5-HT_(1B/1D) (Halazy et al., 1996; Perez et al., 1998a; Perez etal., 1998b) will be synthesized by coupling triptan scaffolds (T) todimeric linkers (D), as shown in FIG. 2.

We have identified an analog of 5-NT, which displayed a two-fold greaterpotency towards MDA-MB-231 breast cancer cells than 5-NT confirming theimportance of the alkoxy substituent at the 5 position of the triptan,as well as other potent compounds (Examples section).

We found using our previously described approach (Akar et al., 2010;Landen et al., 2005; Ozpolat et al., 2007; Ozpolat et al., 2010; Vermaet al., 2008) that a neutral charge nanoliposomal 5-NT formulationprepared from DOPC and Tween-20 was effective at inhibiting tumor growthin an orthotopic model of breast cancer (FIG. 3A). We have used RGD- andfolate-modified LCLsto promote tumor delivery of siRNA(Ozpolat et al.,2010). RGD targets liposomes to αvβ3 integrin receptors, which arehighly expressed in breast tumors (Ozpolat et al., 2010). In someembodiments, the targeted liposome delivery system is composed of 4different lipid components e.g. DOPC, tween-20, DSPE-PEG andDSPE-PEG-RGD. In some embodiments, the extrusion process (Hope et al.,1985) will be used to prepare small unilaminar liposomes of ˜100 nm,which can be confirmed using a light microscope. Based on preliminary invitro experiments using RGD-M-LCL a 4-8-fold greater uptake of siRNAinto cells was achieved compared to passive liposomes.

Several triptan derivatives possessing either novel bulky substituentsat the 5-ring position or possessing a cyclized 3-amino functionality(e.g. frovatriptan) are reported to bind 5-HT_(1B) with higher affinitythan 5-NT (Wilson et al., 2011). We have synthesized a triptanderivative, which possesses a slightly longer 5-alkyl substituent than5-NT and exhibits 2-3 fold greater potency in a viability assay(Examples section).

In studies a number of 5-substituted and cyclic triptan derivatives havebeen made. The folate receptor-alpha (FRα) provides an excellentalternative target for this purpose, because it is a highly selectivetumor marker that is over-expressed in about 90% of breast carcinomas(Garin-Chesa et al., 1993; Holm et al., 1994; Jhaveri et al., 2004; Rosset al., 1994). Most normal cells including breast epithelium do notexpress FRα. More importantly, about 80% of metastases derived fromepithelial cancers express FRα.

In some embodiments, the triptan drug is co-administered with an ED₅₀dose of doxorubicin.

Studies targeting eEF-2K by siRNA in tumors resulted in thedown-regulation of c-Src activity in the tumors. We found in studiesthat the encapsulation of the potential therapeutic drug 5-NT in apassive neutral liposome rendered it effective in animals on atwice-weekly treatment regimen.

We are encouraged that a previous toxicology study found that miceexhibited no weight loss, when treated with a high 50 mg/kg dose ofliposomal 5-NT and given the extensive oral use of triptans in the humanpopulation for the treatment of migraines.

Eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate ofprotein synthesis by inactivating eukaryotic elongation factor 2 (eEF-2)via phosphorylation (Nairn et al., 1985; Ryazanov, 1987). Constitutivelyactivated eEF-2K in human breast cancer has been linked to cellproliferation (Parmer et al., 1998). Glioblastoma and breast cancercells also employ eEF-2K to regulate autophagy—a stress-inducedpro-survival pathway (Cheng et al., 2010; Wu et al., 2006).Additionally, eEF-2K has been implicated in aging (Riis et al., 1993).The mTOR pathway negatively regulates eEF-2K activity viaphosphorylation by p70 S6 kinase (Wang et al., 2001), the cdc2-cyclin Bcomplex (Smith and Proud, 2008) and an additional kinase (Browne andProud, 2004). We made the novel discovery that siRNA-mediateddown-regulation of eEF-2K in breast cancer cells leads to thedown-regulation of critical cell-signaling kinases including mTOR,IGF-1R and Src, while concomitantly inducing apoptosis, and inhibitingcell invasiveness and tumor growth. These observations suggest that mTORis modulated by eEF-2K.

Remarkably, our studies show that a clinically safe triptan,5-nonylytryptamine (5-NT) (Glennon and Dukat, 2010), a potent agonist ofthe 5-HT_(1B/1D) serotonin receptors (Glennon et al., 1996a), exhibitsefficacy in both in vitro and in vivo breast cancer models, with thenotable down-regulation of eEF-2K as well as c-Src/FAK and thedownstream IGF-1R, PI3K/Akt and mTOR signaling pathways. Significantly,we have observed similar downstream effects when eEF-2K isdown-regulated by siRNA, with the inhibition of key protumorigenicsignaling proteins such as c-Src, FAK, IGF-1R, Akt and mTOR. Critically,5-NT exhibits high selectivity for cancer cell lines overnon-tumorigenic cells, and was shown to be non-toxic to mice. Our dataindicates that the targeted down-regulation of eEF-2K by siRNA inducesapoptosis, and inhibits cell proliferation and migration. Additionally,critical signaling proteins including mTOR, IGF-1R and Src aredown-regulated. The 5-HT1B receptor is over-expressed in breast cancercells, and significantly, we found that 5-NT down-regulates eEF-2K andexhibits similar effects to the down-regulation of eEF-2K by siRNA inboth in vitro and in vivo breast cancer models. We have also synthesizeda triptan derivative (KD06) which is more potent than 5-NT.

We made the novel discovery that siRNA-mediated down-regulation ofeEF-2K in breast cancer cells leads to the down-regulation of criticalcell-signaling kinases including mTOR, IGF-1R and Src, whileconcomitantly inducing apoptosis, and inhibiting cell invasiveness andtumor growth. These observations suggest that key signaling pathways aremodulated by eEF-2K.

FIG. 7 indicates that knockdown of eEF-2K inhibits in vitroproliferation of breast cancer cells, colony formation and invasion, andinduces apoptosis. Silencing of eEF-2K by siRNA significantly inhibitsthe number of colonies formed by MDA-MB-231 (FIG. 7C) which indicatesthat eEF-2K promotes cell proliferation (FIG. 7B). In addition,knockdown of eEF-2K inhibits migration/invasion (FIG. 7D). Cleavage ofPARP in MDA-MB-231 cells indicates that eEF-2K knockdown inducesapoptosis. Overexpression of eEF-2K by transfection increases cellproliferation and invasion. FIG. 7E indicates that eEF-2K isoverexpressed across a variety of breast cancer cell lines. The data inFIG. 5 suggests that 5-NT works through the down regulation of eEF-2K.Treatment of MCF-7 cells with 5-NT down-regulates eEF-2K and exhibitssimilar effects as eEF-2K siRNA, including down-regulation of criticalsignaling proteins, inhibition of cell growth and induction ofapoptosis. The data in FIG. 3A suggest that 5-NT potently inhibitsbreast cancer tumor growth in a mouse model. Using nanoliposomaldelivery technology we discovered that the systemic (i.v.)administration of 5-NT at 5.8 mg/kg (twice weekly) significantlyinhibits growth of highly aggressive and metastatic triple negative(Her−, ER−, Pr−) MDA-MB-231 human breast tumors in an in vivo orthotopicxenograft breast cancer model in nude mice. FIG. 7A indicates thateEF-2K siRNA significantly inhibits tumor growth in the MDA-MB-231orthotopic mouse model. DOPC-liposomes containing two differenteEF-2K-targeting siRNAs reduce tumor size significantly in the mousemodel. FIG. 8 indicates that inhibition of eEF-2K blocks activity ofc-Src/FAK and downstream IGF-1R, PI3K/Akt and mTOR signaling pathways.Knockdown of eEF-2K levels down-regulates critical signaling proteinsincluding Src, FAK, IGF-1R, Akt and mTOR. FIG. 8 indicates that eEF-2Kregulates proteins involved in cell cycle progression and angiogenesis.Knockdown of eEF-2K levels affects expression of cell cycle progressionmarkers with increase in p27 and decrease in cyclin D1 levels.Downregulation of eEF-2K also decreases expression of VEGF which isinvolved in angiogenesis.

FIG. 6 indicates that 5-NT inhibits in vitro proliferation of breastcancer cells, colony formation and invasion, and induces apoptosis. 5-NTsignificantly inhibits the number of colonies formed by MCF-7 (FIG. 6C)which indicates that 5-NT inhibits cell proliferation. In addition, 5-NTinduces apoptosis (FIG. 6B) and decreases cell viability (FIG. 6D).Cleavage of PARP in MCF-7 cells (FIG. 9) indicates that 5-NT inducesapoptosis. FIG. 6E indicates that 5-NT exhibits high selectivity forcancer cell lines over non-tumorigenic cells. The data in FIG. 9 suggestthat 5-NT works through the down regulation of eEF-2K. Treatment ofMCF-7 cells with 5-NT down-regulates eEF-2K and exhibits similar effectsas eEF-2K siRNA, including down-regulation of critical signalingproteins, inhibition of cell growth and induction of apoptosis. 5-NTshowed much higher selectivity towards the tumorigenic cell line MCF-7,when compared to MCF-10A, a non-tumorigenic cell line. FIG. 11 suggeststhat 5-NT works through the 5-HT_(1B/1D) receptor. The serotonin5-HT_(1B) receptor is over-expressed in primary, metastatic andresistant breast cancer cells (FIG. 11B). Knockdown of either the5-HT_(1B) or 5-HT_(1D) receptor, blunts the effect of 5-NT (FIG. 11C).5-NT also decreases cAMP levels (FIG. 11D) which is known to beassociated with 5-HT_(1B/1D) receptor signaling.

Using nanoliposomal delivery technology we discovered that the systemic(i.v.) administration of 5-nonylytryptamine (5-NT) (a triptan drug(Glennon et al., 1996)) at 5.8 mg/kg (twice weekly) significantlyinhibits growth of highly aggressive and metastatic triple negative(Her−, ER−, Pr−) MDA-231 human breast tumors in an in vivo orthotopicxenograft breast cancer model in nude mice (FIG. 3A). Most importantly,nanoliposomal 5-NT was shown to be non-toxic to mice and to normalcells. However, it induces apoptosis (FIG. 3B), inhibits colonyformation (FIG. 3C) and inhibits growth (FIG. 3D) of MDA-231 and MCF-7cells.

5-NT is a potent and specific ligand for the serotonin 5-HT_(1B/1D)receptors (Glennon et al., 1996). We also found that 5-NT induced robustapoptosis in MCF-7 cells (FIG. 3B), and induced the down-regulation ofprotein kinases associated with critical survival pathways (e.g. IGF-1R,Akt, mTor, ERK) and/or invasion (e.g. c-Src and Fak) (FIG. 5). Inaddition it down-regulated proteins important for cell cycle progression(e.g. cyclin D) and angiogenesis (e.g. VEGF) (FIG. 5).

While the antagonist SB224289 was found to be quite toxic in mice, 5-NTwas found to potently inhibit breast cancer tumor growth (FIG. 3A)without displaying any toxicity (i.e. weight loss) at a high dose of 50mg/kg.

Targeted delivery of therapeutics and diagnostics to tumor cells andvasculature is recognized as a powerful approach for the treatment ofcancer (Ozpolat et al., 2010). We recently developed a neutralnanoliposome (˜mean diameter 65 nm) composed of neutral-chargedioleylphosphatidylcholine (DOPC), which can target siRNA in vivo intotumor cells 10- and 30-fold more effectively than cationic liposomes ornaked siRNA, respectively (Ozpolat et al., 2010). We effectivelydelivered 5-NT to breast tumors in an orthotopic model using the sameliposome. 5-NT derivatives are generally metabolized efficiently byfirst pass through the liver and have a plasma half-life of only 2-3hours. Significantly, the nanoliposome delivery vehicle requiredadministration of the drug only twice per week at a low 5.8 mg/kg doseto facilitate significant tumor growth inhibition (FIG. 3A).

We discovered that 5-nonylytryptamine (5-NT) (a triptan drug with highspecificity for 5-HT_(1B/1D)) significantly inhibits growth and inducesapoptosis and autophagy in highly aggressive and metastatic Her(−) ER(−)PR(−) in vitro models of human breast cancer. We also found that acombination of 5-NT with a low dose of doxorubicin (a commonchemotherapy) leads to synergistic cell death in MCF-7 cells and that5-NT, when combined with doxorubicin, induces the cell-cycle inhibitor(p27) and the translation inhibitor, programmed cell death 4 (PDCD4). Wediscovered that using nanoliposomal delivery technology the systemic(i.v.) administration of 5-nonylytryptamine (5.8 mg/kg, twice weekly)significantly inhibits growth of tumors in an in vivo orthotopicxenograft breast cancer model in nude mice. We found that nanoliposomal5-NT is non-toxic to mice and normal cells. Our novel neutrally chargedDOPC-based nanovector delivery system has been shown to increasetargeting of molecules such as siRNA in vivo into tumor cells by30-fold. Folate or RGD expressing nanoliposomes exhibit a further 5-9fold enhancement. We found that the 5-HT_(1B) receptor is over-expressedin estrogen receptor ER(−), ER(+), triple negative, highly aggressivemetastatic and drug resistant (tamoxifen and chemotherapy) breastcancers. An antagonist of the 5-HT_(1B) receptor induces apoptotic celldeath, inhibits Bcl-2 expression, as well as the PI3K/Akt/mTOR and STAT3signaling pathways, all of which are associated with progression,metastasis and resistance to most chemotherapeutics, hormone therapy andradiotherapy.

5-NT analogs may be optimally encapsulated in liposomes at a drug tolipid ratio of approximately 1:10 w/w. Drug-lipid compatibility testsmay be performed using differential scanning calorimetry to guide theselection of the optimal lipid composition for entrapment of 5-NTanalogs (Chiu and Prenner, 2011). Liposomal drug formulations may beprepared as follows: Solution A: The RGD-conjugated neutral lipidDSPE-PEG₂₀₀₀-RGD (from Avanti polar lipids Inc.) and DSPE-PEG₂₀₀₀(varied ratio) may be mixed with dioleoylphosphatidylcholine (DOPC) inan overall ratio of ˜1:11; w/w in the presence of ˜5% Tween-20 (w/w).Solution B:DOPC may be mixed with the triptan in a ratio of ˜1:10-20 inthe presence of excess tertiarybutanol. Solution A and B may then bemixed to give a final ratio of ˜(1:10; w/w) drug:lipid. The mixture maythen be frozen and lyophilized overnight. Before incubating with cells,the lyophilized powder may be mixed with saline solution to makeliposomes and extruded through a 100 nm membrane. The amount of eachdrug analog successfully encapsulated may be assessed following themethod of Liu et al. (Liu et al., 2010) where RGD-M-LCL is separatedfrom unloaded drug using a Sephadex-G50 column. Drug release fromRGD-M-LCL may be determined in medium with 50% FBS over 2-120 h usingthe same approach. To evaluate the uptake of liposomes into breastcancer cells, a fluorescent assay may be adopted that we previously usedto characterize liposome uptake (Ozpolat and Lachman, 2003). Followingthe in vitro tests, verification of 5-NT release in vivo may beinvestigated in five control animals with tumors, and plasmaconcentrations may 1 be monitored (0.5-8 h post injection) by HPLC toassess release of the drug following in vivo injections. Tumor and othertissues such as liver, spleen, kidney, heart, lung, small intestine,uterus and muscle may be collected after a single injection of eachformulation. The levels of drug may be determined in tissues andexpressed per gram of tissue weight.

Example 21 Compound Synthesis Chemistry Synthesis of Various TryptamineDerivatives General procedure for the synthesis of3-(2-Aminoethyl)-5-(alkoxy)indole hydrochloride (KD01-KD-09)

A 250 mL round bottom flask was charged with potassium carbonate (650mg, 4.7 mmol) and serotonin hydrochloride salt (500 mg, 2.35 mmol) andstirred in H₂O/CH₃CN (2/1, 10 mL) till all the solid material haddissolved. Di-tert-butyl dicarbonate (564 mg, 2.59 mmol) was added viasyringe, and the resulting yellow solution was allowed to stir at roomtemperature for 24 h. The reaction mixture was diluted with water 10 mland the resulting precipitate was extracted with EtOAc (3×75 mL), andthe combined organic layer was washed with H₂O (1×5 mL) and brine (1×5mL). The EtOAc layer was dried over (Na₂SO4) and solvent evaporatedunder reduced pressure at 25° C. to obtain 610 mg of N-t-BOC-serotoninas brown foam. Yield: 93%, mp 52-54° C. LRMS (ESI): M+ for C₁₅H₂₀N₂O₃,calculated 276.14. found 276.14.

N-t-BOC serotonin (0.5 mmol) and potassium carbonate (1 mmol) wasdissolved in CH₃CN (4 mL) at 25° C. in a 250 mL round bottom flaskfitted with a reflux condenser. Alkyl bromide (0.53 mmol) was added viasyringe and the resulting reaction mixture was heated to 80° C. for 24h. The reaction mixture was allowed to cool to 25° C. and the solventwas evaporated under reduced pressure. The residue obtained was purifiedusing flash chromatography with 20-30% EtOAc/hexanes as eluent. Thealkylated serotonin N-t-BOC serotonin derivatives were obtained as palebrown oil.

The above alkylated serotonin N-t-BOC serotonin derivative (0.11 mmol)was dissolved in EtOAc (0.2 mL) in a 100 mL round bottom flask. To theabove solution was added HCl (3 M) in EtOAc (0.6 mL) and the reactionmixture was allowed to stir for 2 h. A white precipitate formation wasobserved after 10 min. Solvent was evaporated under reduced pressure at25° C. and the product was triturated with anhydrous Et₂O. The solidproduct was collected by filtration and washed well with anhydrous Et₂Oand cold EtOAc to obtain the product as white precipitate.

Scheme 1: Synthesis of 2-(5-alkoxy-1H-indol-3-yl)ethanaminehydrochloride (KD01-KD09)

General procedure for the synthesis ofN-(2-(5-alkoxy-1H-indol-3-yl)ethyl)acetamide (KD10-KD16)

The 3-(2-Aminoethyl)-5-(alkoxy)indole hydrochloride (0.05 mmol) wasdissolved in CH₃CN/H₂O (2/1, 0.5 mL), followed by addition of NaHCO₃(0.25 mmol) in a 5 mL dram vial. To the cloudy white solution was thenadded Ac₂O (0.15 mmol) and the reaction mixture was stirred at 25° C.for 4 h. The reaction mixture wad then diluted with water (4 mL) andextracted with EtOAc (3×2 mL). The EtOAc layer was washed with water(1×2 mL) and brine (1×2 mL) and dried over Na₂SO4. The solvent wasevaporated under reduced pressure at 30° C. to obtain the corresponding3-(2-acetylaminoethyl)-5-(alkoxy)indoles as pale yellow oil whichsolidifies upon standing.

Scheme 2: synthesis of N-(2-(5-alkoxy-1H-indol-3-yl)ethyl)acetamide(KD10-KD16)

Scheme 3: synthesis of2-(5-methoxy-1H-indol-3-yl)-N,N-dimethylethanamine (KD19) and2-(5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)-N,N-dimethylethanamine(KD20)

A reported procedure was followed to prepare the compounds (1).

Scheme 4: synthesis of2-(5-(2-(2-(2-methoxyethoxyl)ethoxy)ethoxy)-1H-indol-3-yl)ethanaminehydrochloride (KD21)

This compound was prepared by the procedure similar to syntheses of2-(5-alkoxy-1H-indol-3-yl) ethanamine hydrochloride.

Procedure for synthesis of2-(5-(nonyloxy)-1H-pyrrolo[3,2-b]pyridin-3-yl)ethanamine (KD25)

The N-phthaloyl protected 5-methoxy-4-azaindole was prepared by heatingthe 5-hydrazinyl-2-methoxypyridine hydrochloride (502 mg, 2.86 mmole)and 2-(4,4-diethoxybutyl)isoindoline-1,3-dione (1 g, 3.43 mmole) in 50mL of 4% sulfuric acid and 8 mL ethanol at 95° C. for 2 h in a 250 mLround bottom flask. The reaction mixture was cooled to 25° C. andneutralized with 30% NH4OH. The reaction mixture was extracted withEtOAc (1×20 mL) and washed with water and brine. The organic layer wasdried over Na2SO4 and solvent evaporated to obtain 820 mg of product aspale brown solid. Yield: 88%.

The N-phthaloyl protected 5-methoxy-4-azaindole (300 mg, 0.94 mmole) wasrefluxed in HBr/CH3CO2H (3 mL) for 48 h in a 100 mL round bottom flaskfitted with a refluxing condensor. The reaction mixture was cooled to25° C. and diluted with water (10 mL). The reaction mixture wasneutralized with 10% NaOH solution and extracted with EtOAc (3×5 mL) andwashed thoroughly with water (2×5 mL) and brine (2×5 mL). The organiclayer was dried over Na2SO4 and solvent evaporated to obtain 190 mg ofproduct, N-phthaloyl protected 5-hydroxy-4-azaindole as pale brownsolid.

N-phthaloyl protected 5-hydroxy-4-azaindole (80 mg, 0.26 mmole) andDIPEA (0.15 mL, 0.63 mmole) was dissolved in 2 mL DMF and stirred at 25°C. for 5 min in a 25 mL round bottom flask. To the reaction mixture wasthen added n-nonyl bromide (60 mg, 0.29 mmole) and reaction continued at110° C. for 24 h. The reaction mixture was cooled to 25° C. and solventremoved under reduced pressure. The residue was purified by flashchromatography eluting with 50% EtOAc/hexanes to afford productN-phthaloyl protected 5-nonyloxy-4-azaindole as a dark yellow solid (31mg, 42%). Rf 0.4 (50% EtOAc/hexanes).

N-phthaloyl protected 5-nonyloxy-4-azaindole (20 mg, 0.05 mmole) andhydrazine (3 mg, 0.09 mmole) was dissolved in CH2Cl2 (0.5 mL) in a 25 mLround bottom flask. EtOH (1 mL) was added to the reaction mixture andallowed to stir overnight at 25° C. for 12 h, after which it was cooledto 0° C., and filtered, and the white solid material was washed with 3mL of cold CH2Cl2. The filtrate was evaporated under reduced pressure toafford 7 mg (50%) of 2-aminoethyl-5-nonyloxy-4-azaindole as a yellowsolid. LRMS (ESI): M+1 for C18H29N3O calcd 304.4. found 304.4.

Scheme 5: synthesis of 4-azaindoles (KD22-KD25)

Scheme 6: Synthesis of7-((3-(2-aminoethyl)-1H-indol-5-yl)oxy)heptanenitrile hydrochloride(KD28)

This compound was prepared by the procedure similar to syntheses of2-(5-alkoxy-1H-indol-3-yl) ethanamine hydrochloride.

Scheme 7: synthesis of tryptamine dimers (KD31-KD32)

These compounds were synthesized following a published procedure (3).

Procedure for the synthesis of N-alkylated derivatives of tryptamine(KD33-KD37)

These compounds were synthesized by a slightly modified reportedprocedure (2). Tryptamine hydrochloride salt (0.8 mmol) was dissolved inCH2Cl2 (10 mL) and cooled to 0° C. in an ice bath in a 100 mL roundbottom flask. Et3N (3.2 mmol) was added dropwsie to the cold solutionfollowed by addition of n-decanoyl chloride (1.6 mmol). The reactionmixture was stirred for 2 h at room temperature and quenched by additionof water (10 mL) and extracted with CH2Cl2 (3×10 mL). The organic layerwas then dried over Na2SO4, filtered and concentrated in vacuo. Theresidue was purified by flash chromatography eluting with 100% hexanesand 20-30% EtOAc/hexanes to afford N-decanoylamide of tryptamine as anyellow solid (236 mg, 92%). R_(f)=0.5 (20% EtOAc/hexanes).

To a solution of above amide (50 mg, 0.16 mmol) in tetrahydrofuran (10mL) was added LiAlH4 (1.59 mL (1 M solution in THF, 1.59 mmol) at 0° C.in a 100 mL round bottom flask. The reaction mixture was refluxed for 4h and then cooled to 25° C. and further to 0° C. in an icebath. Thereaction was quenched with water (10 mL) and extracted with EtOAc (3×5mL). The EtOAc layer was washed with water (1×5 mL), brine (1×5 mL) anddried over Na2SO4. Evaporation of the solvent under reduced pressureafforded the desired product as yellow semisolid (42 mg, 88%).

Scheme 8: synthesis of N-alkylated derivatives of tryptamine andserotonin (KD33-KD37)

A similar procedure was followed to obtain the N-alkylated derivative ofserotonin.

Procedure for the synthesis of2-(5-alkoxy-1H-indol-3-yl)-N,N-dimethylethanamine (KD38-KD46)

The 3-(2-Aminoethyl)-5-(alkoxy)indole hydrogen chloride (0.5 mmol) wasdissolved in MeOH (2.5 mL) along with sodium cyanoboron hydride (0.9mmol), and acetic acid (1.35 mmol) in a 50 mL round bottom flask. Thereaction mixture was stirred for 5 min. to obtain a homogenous solutionand cooled to 0° C. in an ice bath. A solution of 37% formaldehyde (v/vin water) in MeOH (2.5 mL) was added via syringe to the cooled reactionmixture. The temperature was allowed to gradually raise to 25° C. andreaction continued at 25° C. for 12 h. The reaction was quenched withpotassium carbonate (excess) and solvent evaporated. The residueobtained wad resuspended in water (10 mL) and extracted with EtOAc (2×5mL). The EtOAc layer was washed with water (2×5 mL), brine (2×5 mL) anddried over sodium sulfate. The organic layer was evaporated underreduced pressure at 30° C. and dried to obtain the3-(2-N,N-dimethylaminoethyl)-5-(alkoxy)indoles as pale yellow semisolid.

Scheme 9: synthesis of 2-(5-alkoxy-1H-indol-3-yl)-N,N-dimethylethanamine(KD38-KD46)

Procedure for the synthesis of 4- and 7-alkoxy tryptamine (KD48-KD51)

4-Hydroxyindole (500 mg, 3.76 mmol), K2CO3 (1.04 g, 7.51 mmol), andn-nonylbromide (817 mg, 3.94 mmol) was dissolved in acetonitrile (10 mL)and stirred at 25° C. for 10 min in a 250 mL round bottom flask. Thereaction mixture was refluxed for 24 h and cooled to 25° C. The reactionmixture filtered to remove insoluble impurities and solvent evaporatedunder reduced pressure. The residue was purified by flash chromatographyeluting with 100% hexanes and 30% EtOAc/hexanes to afford4-nonyloxyindole as pale yellow solid (847 mg, 87%). R_(f)=0.8 (30%EtOAc/hexanes).

A solution of 4-nonyloxyindole (500 mg, 1.93 mmol),1-dimethylamino-2-nitroethylene (224 mg, 1.93 mmol), and TFA (286 μL) inCH2Cl2 (10 mL) was stirred at 25° C. for 1 h in a 250 mL round bottomflask. The reaction mixture was cooled to 0° C. and quenched withsaturated NaHCO3 solution (10 mL). The organic layer was dried overNa2SO4 and solvent evaporated. The residue thus obtained was purified byflash chromatography eluting with 100% hexanes to 20-30% EtOAc/hexanesto afford 3-(2-nitroethylene)-4-(nonyloxy)indole as a yellow solid (234mg, 37%). R_(f)=0.4 (30% EtOAc/hexanes).

3-(2-nitroethylene)-4-(nonyloxy)indole (230 mg, 0.7 mmol) was dissolvedin THF (6 mL) in 100 mL round bottom flask and cooled to 0° C. in an icebath. LiAlH4 (1 M Solution in THF, 4.2 mL, 4.2 mmol) was added drop wiseto the cooled reaction mixture and the temperature was slowly raised to25° C. The reaction mixture was refluxed for 4 h and cooled to 0° C.followed by quenching with water (10 mL). The reaction mixture wasextracted with EtOAc (3×10 mL) and washed with water (1×10 mL) and brine(1×10 mL), dried over Na2SO4 and filtered. The filtrate was acidifiedwith 3 M HCl in EtOAc (1 mL) and solvent evaporated. The residueobtained was triturated with ether and precipitate obtained filtered andwashed with cold EtOAc and dried to obtain 138 mg of3-(2-aminoethyl)-4-(nonyloxy)indole hydrochloride as a pale yellow solid(138 mg, 58%).

Scheme 10: synthesis of 4- and 7-alkoxy tryptamine (KD48-KD51)

Scheme 11: synthesis of2-(7-alkoxy-1H-indol-3-yl)-N,N-dimethylethanamine (KD and2-(4-alkoxy-1H-indol-3-yl)-N,N-dimethylethanamine

These compounds were synthesized by the procedure similar to2-(5-alkoxy-1H-indol-3-yl)-N,N-dimethylethanamine

Scheme 12: synthesis of N-(2-(4-alkoxy-1H-indol-3-yl)ethyl)acetamide(KD56-KD57) and N-(2-(4-alkoxy-1H-indol-3-yl)ethyl)Acetamide (KD58-KD59)

These compounds were synthesized by the procedure similar toN-(2-(5-alkoxy-1H-indol-3-yl)ethyl)acetamide

General Experimental Methods.

All reactions were carried out under an atmosphere of dry nitrogen.Glasswares were oven-dried prior to use. Unless otherwise indicated,common reagents or materials were obtained from commercial sources andused without further purification. All solvents were dried prior to usewith appropriate drying agents. Dry distilled DMF, DIPEA were obtainedfrom Acros and used as such. Flash column chromatography was performedusing silica gel 60 (230-400 mesh). Analytical thin layer chromatography(TLC) was carried out on Merck silica gel plates with QF-254 indicatorand visualized by UV. ¹H and ¹³C NMR spectra were measured on a VarianDirect Drive 400s, a Varian MERCURY 400, or Varian UNITY+300s andreferenced to Me4Si for ¹H. The purity of the compounds (≧95%) weredetermined using HPLC conducted on an Surveyor 1100 system, using areversed phase C8 column with diode array detection. All LC-MS data werecollected on a Surveyor HPLC (autosampler, quaternary pump, and diodearray detector) and a Thermo LTQ-XL linear ion trap mass spectrometerwith electrospray source. The method of elution used was CH₃CN:H₂O with0.1% formic acid, with a flow rate of 0.5 mL/min. HR electrosprayionization (ESI) mass spectra were recorded on a Waters QTOF Premiermicromass instrument or a Varian 9.4T QFT-ESI ICR system.

¹H (300 MHz DMSO-d₆) δ 10.05 (br, 3H), 7.0 (d, J=9.0 Hz, 2H), 6.89 (d,J=9.0 Hz, 2H), 3.71 (s, 3H)¹³C (100.6 MHz DMSO-d₆) δ 155.23, 139.28,117.56, 114.76, 55.73.

¹H (400 MHz DMSO-d₆) δ 10.84 (br, 1H), 8.12 (br, 2H), 7.25 (d, J=8.8 Hz,1H), 7.19 (d, J=3.8 Hz, 1H), 7.08 (d, J=3.8 Hz, 1H), 6.73 (dd, J=8.8 Hz,1H), 3.99 (t, J=6.4 Hz, 2H), 3.06-2.995 (m, 4H), 1.75-1.67 (m, 2H),1.51-1.42 (m, 2H), 0.95 (t, J=7.2 Hz, 3H); ¹³C (100.6 MHz DMSO-d₆) δ152.89, 131.85, 127.62, 124.39, 112.54, 112.11, 109.64, 101.54, 68.08,31.53, 23.54 (2C), 19.33, 14.26. LRMS (ESI): 233.2 HRMS calculated233.1654. found 233.1650 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.77 (br, 1H), 7.85 (br, 2H), 7.22 (d, J=8.0 Hz,1H), 7.16 (d, J=4.0 Hz, 1H), 7.03 (d, J=4.0 Hz, 1H), 6.71 (dd, J=8.0 Hz,4.0 Hz, 1H), 3.94 (t, J=8.0 Hz, 2H), 3.03-2.99 (m, 2H), 2.95-2.91 (m,2H), 1.75-1.65 (m, 2H), 1.42 (br, 2H), 1.26 (br, 8H), 0.85 (t, J=8.0 Hz,3H); ¹³C (100.6 MHz DMSO-d₆) δ 152.9, 131.86, 127.62, 124.42, 112.54,112.1, 109.59, 101.55, 68.39, 31.71, 29.46, 29.29 (2C), 29.15, 26.13,23.56, 22.55, 14.42. LRMS (ESI): 289.3, HRMS calculated 289.2274 found289.2272 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.76 (br, 1H), 7.86 (br, 2H), 7.21 (d, J=7.9 Hz,1H), 7.15 (d, J=4.0 Hz, 1H), 7.04 (d, J=4.0 Hz, 1H), 6.70 (dd, J=8.0 Hz,4.0 Hz, 1H), 3.93 (t, J=8.0 Hz, 2H), 3.03-2.99 (m, 2H), 2.95-2.91 (m,2H), 1.75-1.65 (m, 2H), 1.42 (br, 2H), 1.26 (br, 10H), 0.85 (t, J=8.0Hz, 3H); ¹³C (100.6 MHz DMSO-d₆) δ 152.9, 131.87, 127.61, 124.46,112.56, 112.1, 109.52, 101.55, 68.39, 31.73, 29.45 (2C), 29.33 (2C),29.13, 26.12, 23.58, 22.55, 14.42. LRMS (ESI) 303.3, HRMS calculated303.2431 found 303.2437 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.78 (br, 1H), 7.92 (br, 2H), 7.21 (d, J=7.8 Hz,1H), 7.16 (d, J=3.9 Hz, 1H), 7.03 (d, J=3.8 Hz, 1H), 6.71 (dd, J=7.8 Hz,4.0 Hz, 1H), 3.93 (t, J=7.9 Hz, 2H), 3.03-2.99 (m, 2H), 2.95-2.91 (m,2H), 1.75-1.65 (m, 2H), 1.41 (br, 2H), 1.24 (br, 12H), 0.84 (t, J=7.9Hz, 3H); ¹³C (100.6 MHz DMSO-d₆) δ 152.9, 131.88, 127.6, 124.47, 112.56,112.09, 109.49, 101.54, 68.38, 31.74, 29.5, 29.43 (2C), 29.32, 29.16,27.31, 26.12, 23.6, 22.55, 14.42. LRMS (ESI) 317.4, HRMS calculated317.25874. found 317.2588 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.77 (br, 1H), 7.82 (br, 2H), 7.22 (d, J=8.1 Hz,1H), 7.16 (d, J=3.9 Hz, 1H), 7.03 (d, J=4.1 Hz, 1H), 6.71 (dd, J=8.2 Hz,4.1 Hz, 1H), 3.93 (t, J=8.2 Hz, 2H), 3.03-2.99 (m, 2H), 2.95-2.91 (m,2H), 1.75-1.65 (m, 2H), 1.41 (br, 2H), 1.24 (br, 14H), 0.84 (t, J=8.1Hz, 3H); ¹³C (100.6 MHz DMSO-d₆) δ 152.75, 131.80, 127.81, 124.04,112.85, 111.72, 109.99, 109.73, 101.65, 68.37, 31.75, 29.51, 29.48 (2C),29.46, 29.32, 29.17, 26.10, 23.62, 22.55, 14.42. LRMS (ESI) 331.3, HRMScalculated 331.2744 found 331.2743 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.73 (br, 1H), 7.85 (br, 2H), 7.17 (d, J=8.2 Hz,1H), 7.11 (d, J=4.2 Hz, 1H), 6.98 (d, J=4.2 Hz, 1H), 6.66 (dd, J=8.2 Hz,4.2 Hz, 1H), 3.88 (t, J=8.1 Hz, 2H), 2.98-2.94 (m, 2H), 2.90-2.86 (m,2H), 1.67-1.62 (m, 2H), 1.41 (br, 2H), 1.24 (br, 16H), 0.78 (t, J=8.0Hz, 3H); ¹³C (100.6 MHz DMSO-d₆) δ 152.8, 131.87, 127.7, 124.46, 112.54,112.09, 109.48, 101.53, 68.39, 31.72, 29.5, 29.48, 29.43 (2C), 29.36,29.32, 29.16, 27.31, 26.12, 23.6, 22.55, 14.42. LRMS (ESI) 345.4, HRMScalculated 345.2900 found 345.2902 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.77 (br, 1H), 7.84 (br, 2H), 7.22 (d, J=8.2 Hz,1H), 7.16 (d, J=4.1 Hz, 1H), 7.03 (d, J=4.1 Hz, 1H), 6.72 (dd, J=8.1 Hz,4.0 Hz, 1H), 3.84 (d, J=4.1 Hz, 2H), 3.01 (br, 2H), 2.95-2.91 (m, 2H),1.70-1.67 (m, 1H), 1.47-1.23 (m, 8H), 0.91-0.85 (m, 6H); ¹³C (100.6 MHzDMSO-d₆) δ 153.17, 131.89, 127.61, 124.49, 112.56, 112.12, 109.51,101.59, 70.99, 30.52, 28.97, 23.88 (2C), 23.58 (2C), 22.99, 14.44,11.45. LRMS (ESI) 289.3, HRMS calculated 289.2274 found 289.2275 (M+1).

¹H (400 MHz DMSO-d₆) δ 10.78 (br, 1H), 7.94 (br, 2H), 7.22 (d, J=7.9 Hz,1H), 7.16 (d, J=3.9 Hz, 1H), 7.03 (d, J=4.0 Hz, 1H), 6.70 (dd, J=7.9 Hz,4.0 Hz, 1H), 3.93 (t, J=6.8 Hz, 2H), 3.06-2.97 (br, 2H), 2.95-2.91 (m,2H), 1.73-1.66 (m, 2H), 1.45-1.36 (m, 2H), 1.27 (br, 20H), 0.83 (t,J=6.8 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 152.90, 131.87, 127.61,124.43, 112.54, 112.09, 109.73, 101.53, 68.38, 31.74, 29.50 (2C), 29.46,29.43, 29.41, 29.40, 29.38, 29.37, 29.33, 29.16, 26.13, 23.57, 22.55,14.42. LRMS (ESI) 373.4, HRMS calculated 373.3213 found 373.3215 (M+1).

¹H (400 MHz DMSO-d₆) δ 10.78 (br, 1H), 8.0 (br, 2H), 7.22 (d, J=8.8 Hz,1H), 7.16 (s, 1H), 7.03 (s, 1H), 6.71 (dd, J=8.6 Hz, 4.3 Hz, 1H), 3.94(t, J=6.4 Hz, 2H), 3.0 (br, 2H), 2.93 (br, 2H), 1.74-1.67 (m, 2H), 1.41(br, 2H), 1.23 (br, 24H), 0.84 (t, J=6.8 Hz, 3H); ¹³C NMR (101 MHz,DMSO-d₆) δ 145.99, 128.83, 126.99, 123.37, 119.55, 111.19, 109.99,103.01, 67.92, 31.75, 29.50 (2C), 29.46, 29.42, 29.38, 29.35, 29.33,29.30, 29.27, 29.21, 29.15, 25.99, 23.64, 22.55, 14.42. LRMS (ESI)401.4, HRMS calculated 401.3526 found 401.3528.

¹H (400 MHZ CDCl₃) δ 7.96 (br, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.0 (d,J=7.9 Hz, 2H), 6.88 (dd, J=7.8 Hz, 4.0 Hz, 1H), 5.52 (br, 1H), 3.99 (t,J=6.8 Hz, 2H), 3.59 (q, J=12.8 Hz, 5.6 Hz, 2H), 2.93 (t, J=6.8 Hz, 2H),1.93 (s, 3H), 1.84-1.77 (m, 2H), 1.48 (br, 2H), 1.32 (br, 8H), 0.89 (t,J=6.0 Hz, 3H); ¹³C (100.6 MHz CDCl₃) δ 170.10, 153.57, 131.48, 127.74,122.69, 113.0, 112.69, 111.87, 101.59, 68.90, 39.68, 31.83, 29.52,29.42, 29.27, 26.15, 25.25, 23.42, 22.67, 14.11. LRMS (ESI) 331.2 (M+1)HRMS calculated 353.2199 found 353.2202 (M+Na⁺).

¹H (400 MHZ CDCl₃) δ 7.95 (br, 1H), 7.26 (d, J=7.8 Hz, 1H), 7.02 (d,J=7.8 Hz, 2H), 6.90 (dd, J=7.9 Hz, 4.0 Hz, 1H), 5.53 (br, 1H), 4.01 (t,J=6.9 Hz, 2H), 3.61 (q, J=12.4 Hz, 5.6 Hz, 2H), 2.95 (t, J=6.8 Hz, 2H),1.94 (s, 3H), 1.86-1.79 (m, 2H), 1.50 (br, 2H), 1.31 (br, 10H), 0.90 (t,J=6.0 Hz, 3H); ¹³C (100.6 MHz CDCl₃) δ 170.10, 153.58, 131.47, 127.75,122.68, 113.02, 112.72, 111.86, 101.59, 68.90, 39.67, 31.88, 29.57,29.52, 29.46, 29.28, 26.14, 25.25, 23.43, 22.67, 14.11. LRMS (ESI) 345.2(M+1), HRMS calculated 367.2356 found 367.2361 (M+Na⁺).

¹H (400 MHZ CDCl₃) δ 7.94 (br, 1H), 7.25 (d, J=7.8 Hz, 1H), 7.02 (d,J=7.8 Hz, 2H), 6.88 (dd, J=7.9 Hz, 4.0 Hz, 1H), 5.52 (br, 1H), 3.99 (t,J=6.9 Hz, 2H), 3.59 (q, J=12.4 Hz, 5.6 Hz, 2H), 2.93 (t, J=6.8 Hz, 2H),1.93 (s, 3H), 1.84-1.77 (m, 2H), 1.48 (br, 2H), 1.27 (br, 12H), 0.88 (t,J=6.0 Hz, 3H); ¹³C (100.6 MHz CDCl₃) δ 170.02, 153.59, 131.47, 127.74,122.67, 113.02, 112.72, 111.86, 101.58, 68.89, 39.67, 31.89, 29.61,29.57, 29.52, 29.46, 29.33, 26.14, 25.25, 23.43, 22.68, 14.12. LRMS(ESI) 359.2 (M+1), HRMS calculated 381.2513 found 381.2515 (M+Na⁺).

¹H (400 MHZ CDCl₃) δ 7.98 (br, 1H), 7.28 (d, J=7.8 Hz, 1H), 7.05 (d,J=7.8 Hz, 2H), 6.90 (dd, J=7.9 Hz, 4.0 Hz, 1H), 5.54 (br, 1H), 4.02 (t,J=6.9 Hz, 2H), 3.62 (q, J=12.4 Hz, 5.6 Hz, 2H), 2.96 (t, J=6.8 Hz, 2H),1.95 (s, 3H), 1.83-1.79 (m, 2H), 1.48 (br, 2H), 1.29 (br, 14H), 0.90 (t,J=6.0 Hz, 3H); ¹³C (100.6 MHz CDCl₃) δ 170.02, 153.58, 131.47, 127.74,126.68, 113.01, 112.70, 111.87, 101.58, 68.90, 39.67, 31.90, 29.62 (3C),29.52, 29.46, 29.34, 26.15, 25.25, 23.42, 22.68, 14.12. LRMS (ESI) 373.2(M+1), HRMS calculated 395.2669 found 395.2672 (M+Na⁺).

¹H (400 MHZ CDCl₃) δ 7.99 (br, 1H), 7.28 (d, J=7.8 Hz, 1H), 7.04 (d,J=7.8 Hz, 2H), 6.90 (dd, J=7.9 Hz, 4.0 Hz, 1H), 5.54 (br, 1H), 4.02 (t,J=6.9 Hz, 2H), 3.62 (q, J=12.4 Hz, 5.6 Hz, 2H), 2.96 (t, J=6.8 Hz, 2H),1.95 (s, 3H), 1.83-1.79 (m, 2H), 1.48 (br, 2H), 1.29 (br, 16H), 0.90 (t,J=6.0 Hz, 3H); ¹³C (100.6 MHz CDCl₃) δ 170.02, 153.58, 131.48, 127.74,122.69, 113.00, 112.70, 111.87, 101.58, 68.90, 39.67, 31.91, 29.67,29.64 (2C), 29.62, 29.52, 29.47, 29.35, 26.15, 25.25, 23.42, 22.68,14.12. LRMS (ESI) 387.3 (M+1), HRMS calculated 409.2823 found 409.2822(M+Na⁺).

¹H (400 MHz CDCl₃) δ 7.94 (br, 1H), 7.26 (d, J=7.8 Hz, 1H), 7.02 (d,J=7.8 Hz, 2H), 6.88 (dd, J=7.9 Hz, 4.0 Hz, 1H), 5.51 (br, 1H), 3.99 (t,J=6.9 Hz, 2H), 3.60 (q, J=12.4 Hz, 5.6 Hz, 2H), 2.93 (t, 2H, J=6.8 Hz),1.93 (s, 3H), 1.82-1.78 (m, 2H), 1.48 (br, 2H), 1.26 (br, 20H), 0.88 (t,J=6.0 Hz, 3H); ¹³C (100.6 MHz CDCl₃) δ 170.02, 153.59, 131.47, 127.75,122.67, 113.02, 112.72, 111.86, 101.58, 68.90, 39.66, 31.91, 29.68,29.65 (3C), 29.62 (2C), 29.52, 29.47, 29.35, 26.15, 25.26, 23.43, 22.69,14.12. LRMS (ESI) 415.3 (M+1), HRMS calculated 437.3139. found 437.3140(M+Na⁺).

¹H (400 MHz CDCl₃) δ 8.01 (br, 1H), 7.26 (d, J=7.8 Hz, 1H), 7.02 (d,J=7.8 Hz, 2H), 6.88 (dd, J=7.9 Hz, 4.0 Hz, 1H), 5.55 (br, 1H), 3.99 (t,J=6.9 Hz, 2H), 3.60 (q, J=12.4 Hz, 5.6 Hz, 2H), 2.94 (t, J=6.8 Hz, 2H),1.93 (s, 3H), 1.82-1.78 (m, 2H), 1.45 (br, 2H), 1.21 (br, 24H), 0.88 (t,J=6.0 Hz, 3H); ¹³C (100.6 MHz CDCl₃) δ 170.12, 153.56, 131.49, 127.74,122.71, 112.99, 112.65, 111.88, 101.58, 68.91, 39.69, 31.92, 29.69 (6C),29.65, 29.63, 29.53, 29.48, 29.36, 26.15, 25.24, 23.40, 22.69, 14.12.LRMS (ESI) 443.3 (M+1), HRMS calculated 465.3452 found 465.3451 (M+Na⁺).

¹H NMR (400 MHz, cdcl₃) δ 7.90 (br, 1H), 7.17 (d, J=7.1 Hz, 1H), 6.96(d, J=2.3 Hz, 1H), 6.93 (s, 1H), 6.79 (dd, J=7.1, 2.3 Hz, 1H), 4.53 (br,1H), 3.93 (t, J=6.6 Hz, 2H), 2.84 (t, J=6.7 Hz, 2H), 1.73 (m, 2H), 1.41(m, 2H), 1.36 (s, 9H), 1.21 (s, 12H), 0.81 (t, J=6.9 Hz, 3H). ¹³C NMR(101 MHz, cdcl₃) δ 155.66, 153.52, 131.63, 127.76, 122.91, 112.83,111.76, 111.24, 101.88, 68.82, 32.08, 29.60, 29.54, 29.49, 29.28, 28.44,26.15, 22.71, 14.12 (2C overlapping). LRMS (ESI) 403.3 (M+1) HRMScalculated 425.2775 found 425.2772 (M+Na⁺).

¹H NMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.7 Hz, 1H), 7.00 (br, 2H), 6.75(d, J=8.7 Hz, 1H), 3.80 (s, 3H), 2.88 (t, J=7.9 Hz, 2H), 2.62 (t, J=7.9Hz, 2H), 2.31 (s, 6H); ¹³C NMR (101 MHz, CD₃OD) δ 153.64, 131.56,127.57, 122.37, 111.87, 111.72, 110.84, 99.64, 59.74, 54.87, 43.66,22.72. LRMS (ESI) 219.2 HRMS calculated 219.1492 found 219.1493 (M+1).

¹H NMR (400 MHz, CD₃OD) δ 7.58 (d, J=8.7 Hz, 1H), 7.21 (d, J=8.7 Hz,1H), 6.52 (d, J=8.0 Hz, 1H), 3.92 (s, 3H), 2.94 (br, 2H), 2.77 (br, 2H),2.37 (s, 6H); ¹³C NMR (101 MHz, CD₃OD) δ 159.70, 141.34, 124.92, 121.48,114.92, 111.73, 103.73, 59.73, 56.30, 43.98, 21.82. LRMS (ESI) 220.2,HRMS calculated 220.1444 found 220.1444 (M+1).

5-Hexaethylene glycol tryptamine ¹H (400 MHZ DMSO-d₆) δ 10.79 (br, 1H),7.87 (br, 2H), 7.23 (d, J=7.8 Hz, 1H), 7.17 (d, J=4.0 Hz, 1H), 7.06 (d,J=4.0 Hz, 1H), 6.73 (dd, J=4.0 Hz, 8.0 Hz, 1H), 4.07 (t, J=5.8 Hz, 2H),3.73 (t, J=5.8 Hz, 2H), 3.58 (m, 2H), 3.50 (m, 4H), 3.41 (m, 2H), 3.21(s, 3H), 2.93 (br, 4H); ¹³C (100.6 MHz DMSO-d₆) δ 152.64, 131.96,127.60, 124.53, 112.61, 112.06, 109.59, 101.64, 71.70, 70.39, 70.26,69.66, 68.07, 66.79, 58.49, 41.70, 23.59. LRMS (ESI) 323.2 HRMScalculated 323.1965 found 323.1967 (M+1)

¹H NMR (400 MHz, CDCl₃) δ 8.05 (s, 1H), 7.73 (br, 4H), 7.48 (d, J=8.6Hz, 1H), 7.16 (s, 1H), 6.51 (d, J=8.6 Hz, 1H), 4.11 (t, J=7.1 Hz, 2H),3.91 (s, 3H), 3.22 (t, J=7.1 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 168.39,159.60, 141.86, 133.68, 132.23, 124.43, 124.26, 122.99, 121.53, 112.84,105.42, 53.03, 38.55, 23.39. LRMS (ESI) 322.2 (M+1), HRMS calculated344.1006 found 344.1004 (M+Na⁺).

¹H NMR (400 MHz, DMSO-d₆) δ 11.08 (br, 1H), 8.05 (br, 2H), 7.70 (d,J=8.7 Hz, 1H), 6.56 (m, J=8.7 Hz, 2H), 3.86 (s, 3H), 3.19 (m, 2H), 2.97(t, J=7.2 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.07, 133.24, 126.68,124.92, 118.37, 110.04, 105.17, 53.18, 23.27 (1C submerged with DMSO-d₆peak). LRMS (ESI) 192.2, HRMS calculated 192.1131 found 192.113 (M+1).

¹H NMR (400 MHz, CD₃OD) δ 7.65 (br, 4H), 7.42 (d, J=7.9 Hz, 1H), 7.08(s, 1H), 6.31 (d, J=7.9 Hz, 1H), 4.04 (t, J=6.6 Hz, 2H), 3.91 (t, J=6.8Hz, 2H), 3.03 (t, J=6.8 Hz, 2H), 1.60 (m, 2H), 1.20 (br, 12H), 0.79 (t,J=6.7 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 168.59, 158.83, 134.12,131.80, 125.24, 122.37, 122.32, 121.24, 111.40, 110.05, 104.29, 65.18,38.55, 31.59, 29.40, 29.21, 29.12, 28.98, 25.90, 23.27, 22.48, 13.27(1C). LRMS (ESI) 434.2, HRMS calculated 434.2438 found 434.2439 (M+1).

¹H NMR (400 MHz, DMSO-d₆) δ 11.06 (br, 1H), 7.93 (br, 2H), 7.68 (d,J=8.7 Hz, 1H), 7.37 (s, 1H), 6.54 (d, J=8.7 Hz, 1H), 4.23 (t, J=6.6 Hz,2H), 3.21-3.14 (m, 2H), 2.96 (t, J=7.2 Hz, 2H), 1.84-1.64 (m, 2H), 1.40(br, 2H), 1.24 (br, 10H), 0.84 (t, J=6.9 Hz, 3H). LRMS (ESI) 304.4, HRMScalculated 304.2383 found 304.2385 (M+1).

¹H (400 MHz DMSO-d₆) δ 10.78 (br, 1H), 7.95 (br, 2H), 7.22 (d, J=8.8 Hz,1H), 7.16 (s, 1H), 7.04 (s, 1H), 6.91 (dd, J=10.8 Hz, 2.0 Hz, 1H); 3.94(t, J=6.4 Hz, 2H), 3.0 (br, 2H), 3.5 (t, J=4.8 Hz, 2H), 2.97-2.93 (m,2H), 1.805-1.67 (m, 4H), 1.29 (br, 10H). ¹³C NMR (101 MHz, DMSO-d₆) δ152.90, 131.87, 127.61, 124.36, 112.84, 112.04, 109.55, 101.65, 68.38,45.98, 35.70, 32.68, 29.44, 29.32, 29.22, 28.52, 27.96, 26.09, 23.57.LRMS (ESI) 381.2, HRMS calculated 381.1536 found 381.1535 (M+1).

¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (s, 1H), 8.03 (br, 2H), 7.22 (d, J=8.7Hz, 1H), 7.16 (d, J=2.1 Hz, 1H), 7.05 (d, J=2.1 Hz, 1H), 6.71 (dd,J=8.7, 2.1 Hz, 1H), 3.93 (t, J=6.4 Hz, 2H), 2.95 (br, 4H), 1.76-1.66 (m,2H), 1.60-1.52 (m, 2H), 1.42 (br, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ153.07, 131.79, 127.80, 124.36, 121.23, 112.85, 111.73, 109.41, 101.41,68.06, 29.03, 28.15, 25.59 (2C), 24.71, 23.59, 16.14. LRMS (ESI) 286.2,HRMS calculated 286.1914 found 286.1915 (M+1).

Acetyltryptamine ¹H (400 MHz CDCl₃) δ 8.01 (br, 1H), 7.54 (d, J=7.9 Hz,1H), 7.32 (d, J=8.2 Hz, 1H), 7.15 (t, J=7.6 Hz, 1H), 7.07 (t, J=7.6 Hz,1H), 6.98 (s, 1H), 5.54 (br, 1H), 3.56-3.52 (m, 2H), 2.92 (t, J=7.2 Hz,2H), 1.86 (s, 3H). ¹³C (100.6 MHz CDCl₃) δ 163.82, 136.37, 127.31,122.26, 122.0, 119.55, 118.69, 112.98, 112.25, 39.87, 25.21, 23.29. LRMS(ESI) 225.1, HRMS calculated 225.0998 found 225.0998 (M+1).

¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s, 1H), 8.01 (br, 2H), 7.27-7.11 (m,1H), 7.05 (d, J=2.0 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.75-6.62 (dd,J=6.3 Hz, 2.0 Hz, 1H), 3.73 (s, 3H), 2.86-2.61 (m, 4H).¹³C NMR (101 MHz,DMSO-d₆) δ 153.29, 131.82, 128.06, 123.69, 112.81, 112.36, 111.37,100.57, 55.76, 43.13, 30.08. LRMS (ESI) 191.2, HRMS calculated 191.1179.found 191.1180 (M+1).

¹H NMR (400 MHz, DMSO-d₆) δ 10.85 (br, 2H), 7.97 (br, 4H), 7.49 (br,4H), 7.25 (d, J=8.7 Hz, 2H), 7.19 (m, 4H), 6.80 (dd, J=8.7, 2.4 Hz, 2H),5.10 (s, 4H), 3.02 (br, 4H), 2.95 (br, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ152.53, 137.59, 132.04, 128.20, 127.60, 124.57, 112.67, 112.26, 109.64,102.14, 70.08, 23.72 (2C overlapping). LRMS (ESI) 455.2, HRMS calculated455.2442 found 455.2446 (M+1).

¹H NMR (400 MHz, DMSO-d₆) δ 10.83 (br, 2H), 8.01 (br, 4H), 7.60 (s, 1H),7.42 (br, 4H), 7.30-7.11 (m, 5H), 6.81 (dd, J=8.7, 2.3 Hz, 2H), 5.12 (s,4H), 2.99 (br, 8H); ¹³C NMR (101 MHz, DMSO-d₆) δ 152.34, 138.38, 132.06,128.87, 127.49, 124.80, 112.85, 112.17, 109.72, 102.05, 70.17, 23.72 (2Coverlapping). LRMS (ESI) 455.2, HRMS calculated 455.2442 found 455.2445(M+1).

¹H NMR (400 MHz, CDCl₃) δ 8.23 (br, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.38(d, J=7.9 Hz, 1H), 7.21 (t, J=7.6 Hz, 1H), 7.13 (t, J=7.6 Hz, 1H), 7.03(s, 1H), 5.56 (br, 1H), 3.61 (q, J=6.4 Hz, 2H), 2.98 (t, J=6.7 Hz, 2H),2.10 (t, J=6.7 Hz, 2H), 1.57 (br, 2H), 1.25 (br, 12H), 0.88 (t, J=6.7Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 173.47, 136.39, 127.33, 122.18,122.02, 119.46, 118.71, 113.01, 111.26, 39.67, 36.90, 31.86, 30.15,29.44, 29.35, 29.28, 25.76, 25.34, 22.67, 14.09. LRMS (ESI) 315.4, HRMScalculated 337.2250 found 337.2251 (M+Na⁺).

¹H (400 MHZ CDCl₃) δ 8.39 (br, 1H), 7.65 (d, J=8.1 Hz, 1H), 7.38 (d,J=8.1 Hz, 1H), 7.21 (t, J=11.9 Hz, 1H), 7.13 (t, J=11.9 Hz, 1H), 7.05(s, 1H), 3.65 (t, J=11.8 Hz, 2H), 3.09-2.96 (br, 4H), 2.66 (t, J=12.0Hz, 2H), 1.26 (br, 2H), 1.29 (br, 12), 0.91 (t, J=12.0 Hz, 3H); ¹³C (101MHz CDCl₃) δ 136.67, 127.61, 122.37, 122.21, 119.47, 119.06, 113.74,111.43, 49.93, 37.75, 33.10, 32.16, 30.0, 29.76, 29.59, 27.58, 26.04,25.61, 22.94, 14.38. LRMS (ESI) 301.2, HRMS calculated 301.2638 found301.2638 (M+1).

¹H NMR (400 MHz, CDCl₃) δ 8.34 (br, 1H), 7.48 (d, J=8.6 Hz, 1H), 7.18(s, 1H), 7.06 (d, J=8.6 Hz, 1H), 5.76 (br, 1H), 3.71 (br, 2H), 3.07 (t,J=6.2 Hz, 2H), 2.73 (t, J=7.6 Hz, 2H), 2.27 (t, J=7.6 Hz, 2H), 2.00-1.88(m, 2H), 1.73 (br, 2H), 1.59 (br, 2H), 1.42 (br, 22H), 1.03 (t, J=6.4Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 176.91, 173.45, 143.87, 134.12,127.56, 123.50, 116.36, 113.17, 112.04, 110.84, 39.64, 36.86, 34.56,33.86, 31.87, 29.46, 29.45, 29.37, 29.31, 29.29, 29.20, 29.13, 25.77,25.25, 25.07, 24.84, 22.67, 14.12. LRMS (ESI) 485.2, HRMS calculated507.3557 found 507.3555 (M+Na⁺).

¹H NMR (400 MHz, CD₃OD) δ 7.14 (d, J=8.7 Hz, 1H), 6.99 (s, 1H), 6.92 (d,J=2.7 Hz, 1H), 6.65 (dd, J=8.7, 2.7 Hz, 1H), 3.44 (t, J=7.2 Hz, 2H),2.85 (t, J=8.0 Hz, 2H), 2.14 (t, J=8.0 Hz, 2H), 1.56 (br, 2H), 1.28 (br,14H), 0.89 (t, J=9.5 Hz, 3H): ¹³C NMR (75 MHz, CDCl₃) δ 150.66, 131.53,128.25, 123.16, 123.03, 112.86, 111.82, 103.46, 49.26, 33.04, 32.15,29.88, 29.82, 29.71, 29.68, 29.58, 29.55, 26.03, 22.93, 14.37. LRMS(ESI) 317.2, HRMS calculated 317.2587 found 317.2591 (M+1).

¹H (400 MHZ CDCl₃) δ 8.11 (br, 1H), 7.17 (d, J=8.0 Hz, 1H), 7.02 (d,J=4.1 Hz, 1H), 6.93 (d, J=4.0 Hz, 1H), 6.80 (dd, J=8.1 Hz, 4.0 Hz, 1H),5.74 (br, 1H), 3.52 (q, J=8.2, 3.8 Hz, 2H), 2.84 (t, J=8.0 Hz, 2H), 2.10(t, J=7.9 Hz, 2H), 1.56 (br, 2H), 1.22 (br, 12), 0.86 (t, J=7.8 Hz, 3H);¹³C (101 MHz CDCl₃) δ 174.11, 150.31, 131.70, 128.26, 123.25, 112.50,112.39, 112.15, 103.43, 39.95, 37.12, 32.11, 29.70, 29.60 (2C), 29.54,26.02, 25.65, 22.92, 14.37. LRMS (ESI) 331.2 (M+1), HRMS calculated353.21995 found 353.22 (M+Na⁺).

¹H (400 MHz CDCl₃) δ 8.47 (br, 1H), 7.52 (d, J=7.7 Hz, 1H), 7.21 (d,J=7.9 Hz, 1H), 7.08 (t, J=7.2 Hz, 1H), 7.02 (t, J=7.2 Hz, 1H), 6.84 (br,1H), 2.87 (t, J=7.8 Hz, 2H), 2.58 (t, J=7.8 Hz, 2H), 2.27 (s, 6H); ¹³C(101 MHz CDCl₃) δ 136.36, 127.42, 121.83, 121.73, 119.09, 118.73,113.90, 111.25, 60.49, 45.37, 23.60. LRMS (ESI) 189.2, HRMS calculated189.1386 found 189.1387 (M+1).

¹H NMR (400 MHz, CD₃OD) δ 7.10 (d, J=8.6 Hz, 1H), 6.93 (s, 1H), 6.86 (d,J=2.3 Hz, 1H), 6.62 (dd, J=8.6, 2.3 Hz, 1H), 2.87 (t, J=7.9 Hz, 2H),2.64 (t, J=7.9 Hz, 2H), 2.29 (s, 6H); ¹³C NMR (101 MHz, CD₃OD) δ 149.98,131.66, 127.91, 122.40, 111.30, 111.24, 110.95, 102.01, 60.03, 43.96,23.14. LRMS (ESI) 205.2, HRMS calculated 205.1335 found 205.1336 (M+1).

¹H (400 MHz CDCl₃) δ 7.93 (br, 1H), 7.24 (d, J=8.8 Hz, 1H), 7.06 (d,J=2.4 Hz, 1H), 7.0 (d, J=2.4 Hz, 1H), 6.86 (dd, J=8.8.0 Hz, 2.4 Hz, 1H),4.01 (d, J=6.8 Hz, 2H), 2.91 (t, J=7.6 Hz, 2H), 2.63 (t, J=7.6 Hz, 2H),2.35 (s, 6H), 1.85-1.78 (m, 2H), 1.50 (br, 2H), 1.33 (br, 8H), 0.90 (t,J=7.2 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 153.39, 131.57, 127.80,122.36, 114.29, 112.85, 111.40, 101.65, 68.70, 60.07, 45.42, 32.22,29.54, 29.35, 29.28, 26.15, 23.59, 22.71, 14.07. LRMS (ESI) 317.3, HRMScalculated 317.2587 found 317.2587 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 7.93 (br, 1H), 7.25 (d, J=8.8 Hz, 1H), 7.08(s, 1H), 7.02 (s, 1H), 6.88 (d, J=8.8 Hz, 1H), 4.03 (t, J=6.7 Hz, 2H),2.93 (t, J=9.6 Hz, 2H), 2.66 (t, J=9.6 Hz, 2H), 2.38 (s, 6H), 1.88-1.78(m, 2H), 1.50 (br, 2H), 1.31 (br, 10H), 0.91 (t, J=6.7 Hz, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 153.39, 131.56, 127.57, 122.04, 113.49, 112.61,111.41, 101.65, 68.95, 60.06, 45.43, 31.89, 29.58, 29.55, 29.49, 29.29,26.46, 23.91, 22.47, 14.07. LRMS (ESI) 331.3, HRMS calculated 331.2744found 331.2744 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 7.94 (br, 1H), 7.25 (d, J=8.7 Hz, 1H), 7.08(s, 1H), 7.02 (s, 1H), 6.88 (d, J=8.7 Hz, 1H), 4.03 (t, J=6.6 Hz, 2H),2.99-2.88 (t, J=9.1 Hz, 2H), 2.68 (t, J=9.1 Hz, 2H), 2.39 (s, 6H),1.89-1.75 (m, 2H), 1.51 (br, 2H), 1.30 (br, 12H), 0.91 (t, J=6.8 Hz,3H); ¹³C NMR (101 MHz, CDCl₃) δ 153.41, 131.54, 127.58, 122.02, 113.51,112.60, 111.43, 101.64, 68.94, 60.06, 45.43, 31.89, 29.59, 29.54, 29.51,29.47, 29.29, 26.46, 23.91, 22.47, 14.07. LRMS (ESI) 345.3, HRMScalculated 345.2900 found 345.2899 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 7.91 (br, 1H), 7.25 (d, J=7.8 Hz, 1H), 7.07(s, 1H), 7.01 (s, 1H), 6.88 (d, J=7.8 Hz, 1H), 4.03 (t, J=6.6 Hz, 2H),2.94 (t, J=9.6 Hz, 2H), 2.66 (t, J=9.6 Hz, 2H), 2.37 (s, 6H), 1.90-1.76(m, 2H), 1.51 (br, 2H), 1.29 (br, 14H), 0.90 (t, J=6.5 Hz, 3H); ¹³C NMR(101 MHz, CDCl₃) δ 153.08, 131.00, 127.56, 122.36, 113.73, 112.84,111.70, 101.96, 69.26, 60.06, 45.10, 31.91, 29.63 (2C), 29.54, 29.49(2C), 29.35, 26.15, 23.59, 22.69, 14.40. LRMS (ESI) 359.4, HRMScalculated 359.3057 found 359.3058 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 7.92 (br, 1H), 7.26 (d, J=8.9 Hz, 2H), 7.07(s, 1H), 7.02 (s, 1H), 6.88 (d, J=8.9 Hz, 1H), 4.03 (t, J=7.4 Hz, 2H),2.96 (t, J=9.6 Hz, 2H), 2.72 (t, J=9.6 Hz, 2H), 2.42 (s, 6H), 1.90-1.75(m, 2H), 1.51 (br, 2H), 1.29 (br, 16H), 0.95-0.86 (t, J=9.6 Hz, 3H); ¹³CNMR (101 MHz, CDCl₃) δ 153.40, 131.55, 127.56, 122.36, 113.48, 112.61,111.72, 101.64, 69.26, 59.74, 45.11, 32.22, 29.68 (2C), 29.64, 29.63,29.55, 29.49, 29.35, 26.16, 23.27, 22.71, 14.07. LRMS (ESI) 373.4, HRMScalculated 373.3213 found 373.3212 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 7.93 (br, 1H), 7.25 (d, J=8.7 Hz, 1H), 7.07(s, 1H), 7.01 (s, 1H), 6.88 (d, J=8.7 Hz, 1H), 4.03 (t, J=6.7 Hz, 2H),2.93 (t, J=10.8 Hz, 2H), 2.67 (t, J=9.6 Hz, 2H), 2.38 (s, 6H), 1.90-1.76(m, 2H), 1.49 (br, 2H), 1.28 (br, 20H), 0.90 (t, J=6.8 Hz, 3H); ¹³C NMR(101 MHz, CDCl₃) δ 153.40, 131.56, 127.80, 122.05, 113.72, 112.60,111.70, 101.65, 68.96, 60.05, 45.09, 31.91, 29.70 (2C), 29.69, 29.66(2C), 29.63, 29.55, 29.50, 29.36, 26.16, 23.91, 22.47, 13.82. LRMS (ESI)401.4, HRMS calculated 401.3526 found 401.3527 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 7.89 (br, 1H), 7.25 (d, J=8.6 Hz, 1H), 7.08(s, 1H), 7.02 (s, 1H), 6.88 (d, J=8.6 Hz, 1H), 4.02 (t, J=7.2 Hz, 2H),2.93 (t, J=9.6 Hz, 2H), 2.66 (t, J=9.6 Hz, 2H), 2.37 (s, 6H), 1.89-1.75(m, 2H), 1.50 (br, 2H), 1.28 (br, 24H), 0.90 (t, J=8.0 Hz, 3H); ¹³C NMR(101 MHz, CDCl₃) δ 153.39, 131.23, 127.80, 122.04, 113.71, 112.61,111.41, 101.97, 68.95, 60.07, 45.42, 32.24, 29.70 (4C), 29.66, 29.63,29.55, 29.50, 29.36, 26.16, 23.58, 22.69, 14.06. LRMS (ESI) 429.4, HRMScalculated 429.3839 found 429.3837 (M+1).

Bisoctatryptaminedecanoyl ¹H (400 MHz CDCl₃) δ 8.29 (br, 2H), 7.26 (d,J=7.8 Hz, 2H), 7.02 (d, J=16.0 Hz, 4H), 6.88 (dd, J=7.6 Hz, 4.0 Hz, 2H),5.56 (br, 2H), 3.99 (t, J=8.0 Hz, 2H), 3.61 (q, J=12.4 Hz, 5.6 Hz), 2.94(t, J=8.0 Hz, 2H), 2.10 (t, J=7.8 Hz), 1.84-1.77 (m, 4H), 1.57 (br, 4H),1.48 (br, 4H), 1.32 (br, 18H), 1.24 (br, 14H), 0.90 (t, J=7.6 Hz, 6H);¹³C (101 MHz CDCl₃) δ 173.13, 153.51, 131.57, 127.69, 122.78, 112.89,112.56, 111.93, 109.99, 101.59, 68.91, 39.39, 36.84, 31.83, 29.53,29.43, 29.28, 29.07, 29.00, 26.16, 25.61, 25.35, 22.67, 14.12. LRMS769.2 (M−1).

4-nonylindole ¹H (400 MHz CDCl₃) δ 8.13 (br, 1H), 7.11 (m, 2H), 7.0 (d,J=7.8 Hz, 1H), 6.68 (t, J=6.0 Hz, 1H), 6.52 (d, 7.8 Hz, 1H), 4.12 (t,J=8.0 Hz, 2H), 1.91 (m, 2H), 1.52 (m, 2H), 1.30 (br, 10H), 0.91 (t,J=8.0 Hz, 3H). LRMS (ESI) 260.2 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.91 (br, 1H), 7.78 (br, 2H), 7.03 (s, 1H), 6.92(m, 2H), 6.43 (d, J=7.2 Hz, 1H), 4.01 (t, J=8.0 Hz, 2H), 3.06 (br, 4H),1.78 (m, 2H), 1.42 (br, 2H), 1.24 (br, 10H), 0.83 (t, J=7.2 Hz, 3H); ¹³C(101 MHz DMSO-d₆) δ 153.61, 138.66, 122.84, 122.55, 116.96, 109.74,105.25, 99.93, 67.54, 31.75, 29.46 (2C), 29.29, 29.23, 29.18, 26.13,25.44, 22.57, 14.43. LRMS (ESI) 303.2, HRMS calculated 303.2431. found303.2429 (M+1).

¹H (400 MHZ DMSO-d₆) δ 10.90 (br, 1H), 7.74 (br, 2H), 7.01 (br, 1H),6.92 (m, 2H), 6.43 (d, J=7.8 Hz, 1H), 4.01 (t, J=8.0 Hz, 2H), 3.06 (br,4H), 1.78 (m, 2H), 1.43 (br, 2H), 1.15 (br, 16H), 0.84 (t, J=8.0 Hz,3H); ¹³C (101 MHz DMSO-d₆) δ 145.99, 128.83, 126.99, 123.36, 119.54,111.19, 110.36, 109.99, 103.00, 67.91, 31.75, 29.52 (2C), 29.49, 29.47,29.31 (2C), 29.17, 25.98, 23.63, 22.55, 14.41. LRMS (ESI) 345.2, HRMScalculated 345.2900 found 345.2899 (M+1).

¹H (400 MHz DMSO-d₆) δ 10.91 (br, 1H), 8.0 (br, 2H), 6.89 (t, J=8.0 Hz,1H), 6.62 (d, J=7.2 Hz, 1H), 7.1 (s, 1H), 7.12 (br, 1H), 4.1 (t, J=6.8Hz, 2H), 3.0 (br, 2H), 2.98-2.93 (m, 2H), 1.80-1.72 (m, 2H), 1.50-1.43(m, 2H), 1.25 (br, 10H), 0.84 (t, J=6.8 Hz, 3H); ¹³C NMR (101 MHz,DMSO-d₆) δ 152.89, 131.87, 127.63, 124.35, 112.54, 112.08, 109.71,101.53, 68.38, 31.74, 29.49, 29.46, 29.33, 29.16, 26.13, 23.90, 22.55,14.42. LRMS (ESI) 303.2, HRMS calculated 303.2431 found 303.2430 (M+1).

7-dodecaoxy tryptamine ¹H (400 MHZ DMSO-d₆) δ 10.90 (br, 1H), 8.03 (br,2H), 7.11 (m, 2H), 6.68 (t, J=7.9 Hz, 1H), 6.61 (d, J=7.9 Hz, 1H), 4.07(t, J=8.0 Hz, 2H), 2.98 (br, 4H), 1.76 (m, 2H), 1.46 (br, 2H), 1.25 (br,16H), 0.83 (t, J=8.0 Hz, 3H); ¹³C (101 MHz DMSO-d₆) δ 145.99, 128.83,126.99, 123.36, 119.54, 111.19, 110.36, 109.99, 103.00, 67.91, 31.75,29.52 (2C), 29.49, 29.47, 29.31 (2C), 29.17, 25.98, 23.63, 22.55, 14.41.LRMS (ESI) 345.2, HRMS calculated 345.2900 found 345.2901 (M+1).

4-nonylindole-3-nitroethylene ¹H (400 MHz CDCl₃) δ 8.64 (br, 1H), 8.56(d, J=16.0 Hz, 1H), 8.06 (d, J=16.0 Hz, 1H), 7.60 (br, 1H), 7.24 (t,J=7.9 Hz, 1H), 7.05 (d, J=8.0 Hz, 1H), 6.69 (d, J=7.9 Hz, 1H), 4.18 (t,J=8.0 Hz, 2H), 1.99 (m, 2H), 1.57 (br, 2H), 1.30 (br, 10H), 0.90 (t,J=8.0 Hz, 3H). LRMS (ESI) 331.2 (M+1).

¹H (400 MHz CDCl₃) δ 8.10 (br, 1H), 7.07 (t, J=7.9 Hz, 1H), 6.95 (d,J=7.8 Hz, 1H), 6.90 (s, 1H), 6.50 (d, J=7.9 Hz, 1H), 4.10 (t, J=6.6 Hz,2H), 3.12 (t, J=8.2 Hz, 2H), 2.71 (t, J=8.2 Hz, 2H), 2.37 (s, 6H), 1.98(m, 2H), 1.52 (br, 2H), 1.23 (br, 10H), 0.91 (t, J=6.6 Hz, 3H); ¹³C (101MHz CDCl₃) δ 154.16, 138.06, 122.68, 120.38, 117.29, 114.68, 104.17,99.83, 67.67, 61.41, 45.26, 31.88, 29.61 (2C), 29.49, 29.46, 29.30,24.94, 22.67, 14.11. LRMS (ESI) 331.2, HRMS calculated 331.2744 found331.2747 (M+1).

¹H (400 MHz CDCl₃) δ 8.02 (br, 1H), 7.05 (t, J=7.8 Hz, 1H), 6.91 (d,J=7.8 Hz, 1H), 6.91 (s, 1H), 6.50 (d, J=7.8 Hz, 1H), 4.09 (t, J=6.7 Hz,2H), 3.08 (t, J=8.1 Hz, 2H), 2.70 (t, J=8.1 Hz, 2H), 2.35 (s, 6H), 1.89(m, 2H), 1.56 (br, 2H), 1.29 (br, 16H), 0.91 (t, J=6.6 Hz, 3H); ¹³C (101MHz CDCl₃) δ 154.19, 137.54, 123.80, 122.68, 120.36, 114.68, 102.53,99.97, 67.49, 61.49, 45.42, 31.91, 29.65 (3C), 29.47, 29.34 (2C), 29.30,24.85, 24.44, 22.69, 14.39. LRMS 373.4, HRMS calculated 373.3213 found373.3213 (M+1).

¹H NMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 7.12 (d, J=8.0 Hz, 1H),6.98-6.90 (m, 2H), 6.56 (d, J=7.6 Hz, 1H), 4.04 (t, J=6.5 Hz, 2H), 2.87(t, J=8.0 Hz, 2H), 2.59 (t, J=8.0 Hz, 2H), 2.29 (s, 6H), 1.83-1.72 (m,2H), 1.42 (br, 2H), 1.21 (br, 10H), 0.82 (t, J=6.9 Hz, 3H); ¹³C NMR (101MHz, CDCl₃) δ 145.88, 128.67, 126.93, 120.90, 119.48, 114.27, 111.40,102.84, 67.81, 60.30, 48.54, 45.10, 31.91, 29.57, 29.43, 29.28, 26.16,23.59, 22.71, 14.06. LRMS (ESI) 331.2, HRMS calculated 331.2744 found331.2742 (M+1).

¹H NMR (400 MHz, CDCl₃) δ 8.17 (br, 1H), 7.20 (d, J=5.6 Hz, 1H),7.04-6.97 (m, 2H), 6.62 (d, J=7.4 Hz, 1H), 4.11 (t, J=6.4 Hz, 2H), 2.93(t, J=6.8 Hz, 2H), 2.64 (t, J=6.8 Hz, 2H), 2.35 (s, 6H), 1.87-1.81 (m,2H), 1.48 (br, 2H), 1.26 (br, 16H), 0.88 (t, J=6.8 Hz, 3H); ¹³C NMR (101MHz, CDCl₃) δ 144.20, 128.64, 126.91, 120.66, 119.34, 114.23, 110.92,103.86, 67.81, 59.78, 48.53, 44.92, 32.02, 29.56 (2C), 29.33 (2C),29.23, 26.12, 23.54, 22.72, 14.29 (1C missing). LRMS (ESI) 373.4, HRMScalculated 373.3213 found 373.3212 (M+1).

N-acetyl-4-nonylindole ¹H (400 MHz CDCl₃) δ 8.20 (br, 1H), 7.10 (t,J=8.0 Hz, 1H), 6.99 (d, J=8.1 Hz, 1H), 6.90 (s, 1H), 6.51 (d, 1H, J=8.0Hz), 5.96 (br, 1H), 4.12 (t, J=6.6 Hz, 2H), 3.60 (m, 2H), 3.10 (t, J=6.4Hz, 2H), 1.89 (s, 3H), 1.52 (br, 2H), 1.31 (br, 12H), 0.91 (t, J=7.6 Hz,3H); (101 MHz CDCl₃) δ 170.07, 153.78, 138.22, 122.90, 121.21, 117.26,113.50, 104.56, 100.12, 67.78, 41.29, 31.86, 29.57 (2C), 29.42, 29.28(2C), 26.26, 23.31, 22.68, 14.11. LRMS (ESI) 367.2, HRMS calculated367.2356 found 367.2355 (M+1).

¹H NMR (400 MHz, CDCl₃) δ 8.02 (br, 1H), 7.08 (t, J=8.0 Hz, 1H), 6.97(d, J=8.0 Hz, 1H), 6.90 (s, 1H), 6.49 (d, J=8.0 Hz, 1H), 5.90 (br, 1H),4.09 (t, J=6.5 Hz, 2H), 3.62-3.54 (t, J=6.3 Hz, 2H), 3.08 (t, J=6.3 Hz,2H), 1.87 (br, 5H), 1.54-1.47 (m, 2H), 1.43-1.35 (m, 2H), 1.26 (br,14H), 0.88 (t, J=6.8 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 170.03, 153.64,138.13, 122.92, 121.25, 117.48, 113.72, 104.53, 100.21, 68.06, 41.09,32.23, 29.66 (3C), 29.63, 29.42 (2), 29.34, 26.26, 23.26, 22.64, 14.17.LRMS (ESI) 409.4, HRMS calculated 409.2826 found 409.2823 (M+1).

¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.11 (d, J=8.6 Hz, 1H),7.01-6.92 (m, 2H), 6.58 (d, J=7.6 Hz, 1H), 5.44 (br, 1H), 4.05 (t, J=6.5Hz, 2H), 3.54-3.49 (m, 2H), 2.88 (t, J=6.6 Hz, 2H), 1.84 (s, 3H),1.83-1.72 (m, 2H), 1.48-1.36 (m, 2H), 1.22 (br, 10H), 0.82 (t, J=6.8 Hz,3H); ¹³C NMR (101 MHz, CDCl₃) δ 170.02, 145.86, 128.68, 126.92, 121.45,119.80, 113.49, 111.15, 102.84, 68.04, 39.99, 31.88, 29.57, 29.43,29.36, 29.28, 26.16, 25.39, 23.39, 22.67, 14.09. LRMS (ESI) 367.2, HRMScalculated 367.2356 found 367.2355 (M+1).

¹H NMR (400 MHz, CDCl₃) δ 8.21 (br, 1H), 7.11 (d, J=8.0 Hz, 1H),7.00-6.91 (m, 2H), 6.58 (d, J=7.5 Hz, 1H), 5.45 (br, 1H), 4.05 (t, J=6.5Hz, 2H), 3.54-3.49 (m, 2H), 2.88 (t, J=6.4 Hz, 2H), 1.84 (s, 3H),1.83-1.72 (m, 2H), 1.43 (br, 2H), 1.30 (br, 2H), 1.20 (br, 14H), 0.81(t, J=6.9 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 170.27, 145.64, 128.68,126.92, 121.25, 119.48, 113.17, 111.15, 102.85, 68.05, 39.98, 31.91,29.67, 29.63, 29.62, 29.43, 29.35, 29.35, 26.46, 25.26, 23.59, 22.69,14.06. LRMS (ESI) 409.2, HRMS calculated 409.2823 found 409.2825 (M+1).

Example 22 Compound Characterization

Once tumors are established (at least 3-5 mm), mice (10 mice/group) willbe given the liposomal drug twice weekly (i.v. injections of drug orcontrol). The treatment will be carried out for at least 3-4 weeks. Whenthe maximum size of local tumors is ˜1-1.5 cm³ in mean value mice willbe euthanized by CO₂ inhalation. Animals will be monitored daily foradverse effects and evidence of toxicity, e.g. weight loss and reduceddaily activity. Mice that have lost 20% of their original weight,exhibit labored breathing, hunched posture, or are incapacitated bytumor burden will be euthanized. The size of the breast cancer tumorxenografts will be measured before and after treatment every week bydirect measurement using an electronic caliper. Imaging of luciferasemodels will occur weekly by measuring bioluminescence using acryogenically cooled IVIS imaging system coupled with data acquisitioncontrolled by a computer running Livinglmage software (Xenogen, Alameda,Calif.). Statistical analysis will be performed. The expression ofregulators of angiogenesis (e.g. VEGF, HIF-1α, CD31), the expression ofelements of endoplasmic stress (phospho-eIF2α), stress pathways (e.g.MAPK) and induction of apoptopsis (PARP, caspase 9 and caspase 3cleavage) will be assessed by western blot analysis. We previouslyoptimized conditions for western blotting of samples from in vivoexperiments (Akar et al., 2008; Ozpolat et al., 2008). Apoptosis willalso be assessed by Annexin V, by examining cell cycle analysis(sub-G1). Evaluation of autophagy will achieved by detection of acidicvesicular organelles, and LC3-II (e.g. FIG. 5), and Beclin-1, expression(Akar et al., 2008). Proliferation will be assessed by the Ki-67proliferation marker. Models:Nude mice (nu/nu) will be obtained fromJackson laboratories. Three models will be used: an MCF-7 (ER+)orthotopic model (Kim and Price, 2005) will be prepared by priming micewith 17β-Estradiol applied subcutaneously (1.7 mg estradiol/pellet)under the left shoulder to promote tumor growth. In this model themammary fat pad under the nipple of each mouse is inoculated (0.1 mL,7×10⁶ breast cancer cells in PBS) by subcutaneous injection using asterile 28-g needle. An MDA-MB-231 orthotopic model (Akar et al., 2010)or luciferase-expressing MDA-MB-231 orthotopic model (Akar et al., 2010)will be prepared in a similar manner by injecting 1 or 2×106 tumorcells, respectively into the mammary fat pad.

IC50 concentrations of compounds were determined by Cell Viability assay(aka proliferation assay, in this case MTS assay was used. MTS, MTT, XTTall similar asays often used as cell viability/toxixty assay). Cellviability in response to compounds such as 5NT and KD06 was studied witha tetrazolium/formazan based assay (MTS, Celltiter 96 aqueous onesolution cell proliferation assay, Promega, Wis.) [Akar U, Chaves-ReyezA, Barria M, Tani A, Sanguino A, Kondo Y, Kondo S, Arun B,Lopez-Berestein G, Ozpolat B: Silencing of Bcl-2 expression by smallinterfering RNA induces autophagic cell death in MCF-7 breast cancercells. Autophagy 2008, 4:669-679, Ozpolat 2007 MCR]. Cells were seededin 96 wells plates at a density of 1.5 to 3.0×10³ cells per well in 100μl of medium. Next day the medium was removed and cells were treatedwith the drug in 100 μl of medium for 72 hours. Plates were read at 490nm wavelength in an Elisa plate reader (Kinetic Microplate Reader,Molecular Devices Corporation, Sunnyvale, Calif.).

Example 23 Autophosphorylation of eEF-2K

The following five autophosphorylation sites were identified, Thr-348,Thr-353, Ser-445, Ser-474 and Ser-500. Mutation of each site to eitherAla or Asp suggested that only Thr-348 is required for activity ofeEF-2K against a peptide substrate in the presence of Ca²⁺/CaM.Stoichiometric analysis indicates the incorporation of ˜3.5 molphosphate/mol enzyme over 3 hours, with the rapid intramolecularincorporation of ˜1 mol phosphate within the first 10 min—this initialphosphate most likely being incorporated at Thr-348.

Cellular homeostasis demands a controlled balance between proteinsynthesis and protein degradation. Eukaryotes regulate their rate ofprotein synthesis through a variety of pathways; several of whichinclude phosphorylation of translation initiation and elongation factors(Hershey, J. W. B. Annual Review of Biochemistry 1991: 60, 717-755;Morley, S. J., and Thomas, G. Pharmacology & Therapeutics 1991: 50,291-319; Proud, C. G. Curr Top Cell Regul 1992: 32, 243-369; Rhoads, R.E. Journal of Biological Chemistry 1999: 274, 30337-30340). An importantcomponent of this regulatory process is the eukaryotic elongation factor2 kinase (eEF-2K). eEF-2K functions to generally impede the elongationphase of translation, thereby disrupting proteins synthesis (Nairn, A.C., et al., Proc Natl Acad Sci USA 1985: 82, 7939-7943; Nairn, A. C.,and Palfrey, H. C. Journal of Biological Chemistry 1987: 262,17299-17303; Ryazanov, A. G. FEBS Letters 1987: 214, 331-334; Ryazanov,A. G., et al. Biochimie 1988: 70, 619-626; Carlberg, U., et al. EuropeanJournal of Biochemistry 1990: 191, 639-645). Additionally, it may alsoinduce the translation of specific transcripts (Weatherill, D. B., etal. Journal of Neurochemistry 2011: 117, 841-855). eEF-2K inhibitstranslation by phosphorylating, and thereby blocking, the ability ofelongation factor 2 (eEF-2) to bind the ribosome (Nairn, A. C., et al.,Proc Natl Acad Sci USA 1985: 82, 7939-7943; Nairn, A. C., and Palfrey,H. C. Journal of Biological Chemistry 1987: 262, 17299-17303; Ryazanov,A. G. FEBS Letters 1987: 214, 331-334; Ryazanov, A. G., et al. Biochimie1988: 70, 619-626; Carlberg, U., et al. European Journal of Biochemistry1990: 191, 639-645). eEF-2 is responsible for the ribosomaltranslocation of the nascent peptide chain from the A-site to the P-siteduring translation (Moldave, K. Annual Review of Biochemistry 1985: 54,1109-1149; Moazed, D., and Noller, H. F. Nature 1989: 342, 142-148;Proud, C. G. Mol Biol Rep 1994: 19, 161-170).

eEF-2K is classified as a Ca²⁺/CaM-dependent protein kinase (CaMK-III)(Nairn, A. C., et al., Proc Natl Acad Sci USA 1985: 82, 7939-7943;Ryazanov, A. G., et al. Biochimie 1988: 70, 619-626; Mitsui, K., et al.Journal of Biological Chemistry 1993: 268, 13422-13433; Redpath, N. T.,and Proud, C. G. European Journal of Biochemistry 1993: 212, 511-520)because it requires Ca²⁺ and calmodulin (CaM) for autophosphorylation.When the cell is in the resting state, basal levels of intracellularcalcium (50-100 nM) appear to be insufficient for CaM to exert an effecton the Ca²⁺/CaM-dependent protein kinase (Chin, D., and Means, A. R.Trends in Cell Biology 2000: 10, 322-328; Swulius, M., and Waxham, M.Cellular and Molecular Life Sciences 2008: 65, 2637-2657). However,during signaling events, influx of calcium into the cytoplasm raises thefree intracellular Ca²⁺ concentration to ˜10-100 μM which is adequatefor the activation of calmodulin (Chin, D., and Means, A. R. Trends inCell Biology 2000: 10, 322-328; Swulius, M., and Waxham, M. Cellular andMolecular Life Sciences 2008: 65, 2637-2657). Autophosphorylation ofeEF-2K in the presence of Ca²⁺/CaM activates the kinase (Mitsui, K., etal. Journal of Biological Chemistry 1993: 268, 13422-13433; Redpath, N.T., and Proud, C. G. European Journal of Biochemistry 1993: 212,511-520).

A compelling factor behind deciphering the mechanism of activation andregulation of eEF-2K is its recent implication in enhancing tumorsurvival (Bagaglio, D. M., and Hait, W. N. Cell Growth Differ 1994: 5,1403-1408; Parmer, T. G., et al. Br J Cancer 1998: 79, 59-64; Arora, S.,et al., Cancer Res 2003: 63, 6894-6899; Hait, W. N., et al., Autophagy2006: 2, 294-296; Wu, H., et al. Cancer Research 2006: 66, 3015-3023).Results reveal that glioblastoma cells employ the kinase to regulateautophagy—a pro-survival pathway stimulated in response to nutrientdeficiency and cell stress (Hait, W. N., et al., Autophagy 2006: 2,294-296; Wu, H., et al. Cancer Research 2006: 66, 3015-3023; Wu, H., etal. Cancer Research 2009: 69, 2453-2460). Cancer research on squamouscarcinoma cells and metastatic breast cancer cells has indicated thattreatment of the malignant cells with chemotherapeutic agents appears toup-regulate the activity of the kinase—an event that has been associatedwith the induction of autophagy (Ren, H., et al., Cancer Res 2005: 65,5841-5847; Dalby, K. N., et al. Autophagy 2010: 6, 322-329). Triggeringof this adaptive mechanism consequently endows the cells with resistanceagainst anti-tumorous agents, and as a result reduces therapeuticpotency (Wu, H., et al. Cancer Research 2009: 69, 2453-2460, Dalby, K.N., et al. Autophagy 2010: 6, 322-329). Additionally, we have foundevidence that eEF-2K promotes proliferation, migration and invasion ofbreast cancer cells (Tekedereli, I., et al, (2011 Submitted)Calmodulin-Dependent Protein Kinase III (Elongation Factor-2 Kinase)Regulates Proliferation and Invasiveness of Breast Cancer Cells). Thesefindings suggest that eEF-2K may be a target for anti-cancer therapy.

This study reports five novel autophosphorylation sites in eEF-2K, whichinclude Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500. Of these, onlyThr-348 is found to be essential for kinase activity against a peptidesubstrate.

Peptide synthesis—For the kinetic analysis of eEF-2K, the peptidesubstrate, Acetyl-RKKYKFNEDTERRRFL-Amide (SEQ ID NO:6) (2,227.8 Da), wassynthesized and purified at the UT Molecular Biology Core Facilities asdescribed earlier (Abramczyk, O., et al. (2011) Purification andcharacterization of tagless recombinant human elongation factor 2 kinase(eEF-2K) expressed in Escherichia coli, Protein Expression andPurificationIn Press, Uncorrected Proof).

General kinetic assays—eEF-2 kinase activity was assayed at 30° C. inBuffer D (25 mM HEPES (pH 7.5), 2 mM DTT, 0.15 μM BSA, 100 μM EDTA, 100μM EGTA, 250 μM CaCl₂, 2 μM calmodulin and 10 mM MgCl₂), containing 150μM (Acetyl-RKKYKFNEDTERRRFL-Amide) (SEQ ID NO:6) peptide substrate(Pep-S), 2 nM eEF-2K enzyme and 0.5 mM [γ-³²P]ATP (100-1000 cpm/pmol) ina final reaction volume of 100 μL. The reaction mixture was incubated at30° C. for 10 min before the reaction was initiated by addition of 0.5mM [γ-³²P] ATP. At set time points, 10 μL aliquots were taken andspotted onto P81 cellulose filters (Whatman, 2×2 cm). The filter paperswere then washed thrice in 50 mM phosphoric acid (15 min each wash),once in acetone (15 min) and finally dried. The amount of labeledpeptide associated with each paper was determined by measuring the cpmon a Packard 1500 scintillation counter.

(Autophosphorylation assay—Autophosphorylation of eEF-2K was carried outin Buffer E (25 mM HEPES (pH 7.5), 2 mM DTT, 0.6 μM BSA, 6 μM CaM, 150μM CaCl₂ and 10 mM MgCl₂) containing 200 nM eEF-2K enzyme and 0.5 mM[γ-³²P]ATP (100-1000 cpm/pmol) in a final volume of 200 μL. The reactionmixture was incubated at 30° C. for 10 min before the reaction wasinitiated by addition of 0.5 mM [γ-³²P]ATP. Aliquots (2 pmol) of eEF-2Kwere removed at intervals over a 3 h time period and the reactionquenched by addition of SDS-PAGE sample loading buffer (125 mM tris-HCl(pH 6.75), 20% glycerol (v/v), 10% 2-mercaptoethanol (v/v), 4% SDS and0.02% bromophenol blue) followed by heating for 10 min at 95° C. Thesamples were resolved by SDS-PAGE and stained with Coomassie BrilliantBlue. Gels were exposed for 1 h in a Phosphorimager cassette which wasthen scanned in a Typhoon Phosphorimager and then analyzed usingImageQuant™ TL software. To determine the stoichiometry of theautophosphorylation, the gels were dried, the pieces containing eEF-2Kexcised, and the associated radioactivity measured with a Packard 1500liquid scintillation analyzer. The mechanism of autophosphorylation wasanalyzed using Buffer F (25 mM HEPES (pH 7.5), 2 mM DTT, 0.6 μM BSA, 0.6μM CaM, 1.5 mM CaCl₂ and 10 mM MgCl₂) containing 0.5 mM [γ-³²P]ATP andvarying concentrations of the purified enzyme (0-400 nM). The reactionwas carried out under conditions in which linear incorporation of ³²Pwas achieved (10 min incubation) and quenched by addition of hotSDS-PAGE sample loading buffer. The extent of phosphate incorporationfor each sample was determined as described above, and then plotted as afunction of enzyme concentration.

Phosphopeptide analysis—Phosphorylation sites on the purified enzymewere detected by mass spectrometry analysis of the in-gel tryptic andchymotryptic digests. The eEF-2K sample (5 μM) purified from E. coli wasresolved using SDS-PAGE (Bio-Rad Ready Gel Tris-HCl Gel 10% precastpolyacrylamide gel), stained with Coomassie Brilliant Blue anddestained. The band corresponding to eEF-2K was carefully excised anddiced into small pieces which were dehydrated twice with 100 μLacetonitrile (ACN) for 5-10 min. Gel pieces were destained further with100 μL of destaining solution (50% methanol and 5% glacial acetic acid)for 1 h, following which the sample was again dehydrated twice with 100μL ACN for 5-10 min. The gel was dried in a speed vacuum (5-10 min) andthe enzyme then reduced with 100 μL 10 mM DTT at 60° C. for 1 h. Theexcess solution was removed and the sample treated with 100 μL ACN for5-10 min. Alkylation was then performed for 1 h in the dark by theaddition of 100 μL 55 mM iodoacetamide. The excess solution was againremoved and the sample treated this time with 50 mM ammonium bicarbonate(pH 8.5) for 5-10 min, dehydrated with 100 μL ACN (5-10 min) and driedin a speed vacuum (5-10 min). The gel pieces were treated with Trypsin(Promega) or Chymotrypsin (Sigma) in 50 mM ammonium bicarbonate (pH 8.5)to give a final protease-enzyme ratio of ˜1:5. Digestion was carried outat 37° C. for 16 h, after which the sample was treated with 5 μL of asolution containing 60% ACN and 5% formic acid, and sonicated for 5 min.Excess solution was removed and retained. ACN (10 μL) was added twiceand the excess solution each time was pooled with the solution from theprevious step. The combined digested peptide sample was dried in a speedvacuum (5-10 min) and stored at −20° C. (The protocol followed wassuggested by the Mass Spectrometry Facility, Department of Chemistry andBiochemistry, UT Austin). To detect potential autophosphorylation sites,5 μM of the purified enzyme was allowed to autophosphorylate in thepresence of CaM, Ca²⁺ and Mg₂ATP for 3 h as outlined above. The samplewas then resolved by SDS-PAGE and the autophosphorylated eEF-2Ksubjected to tryptic and chymotryptic in-gel digestion as described.Tryptic and chymotryptic eEF-2K peptide digests were solubilized inmobile phase A (MPA: 0.1% formic acid and 0.01% trifluoroacetic acid)and adjusted to pH<3 with addition of 10% trifluoroacetic acid. Thepeptides (20 pmol) were then loaded onto a reversed phase trap column(Symmetry C18, 180 μm×20 mm, Waters Corporation) mounted on ananoAcquity UPLC system (Waters Corporation) and washed for 10 min with10 μL/min MPA. A reversed phase nano-LC analytical column (AtlantisdC18, 75 μm×150 mm, Waters Corportation) was then incorporated into theflow path. Peptides were eluted with a linear 30 min 2-30% mobile phaseB (MPB: acetonitrile, 0.1% formic acid and 0.01% trifluoroacetic acid)gradient followed by a linear 10 min 30-60% MPB gradient. Elutedpeptides were infused directly into a Q-TOF Premier mass spectrometer(Waters Corporation) using a nanospray ion source. Mass spectra wereacquired using a nanospray voltage of 3.5 kV, sampling cone voltage of40 V, cone gas (nitrogen) flow of 20 L/h, a source temperature of 100°C., and a collision gas (nitrogen) pressure of 5.1 e⁻³. All mass spectrawere collected for 1.95 s over m/z 50-2000 with an interscan delay of0.1 s. The mass spectrometer was programmed to perform an experimentsequence consisting of an MS analysis at a collision energy of 5.0 V,followed by MS/MS analysis on the 3 most intense ions observed in the MSspectrum. For each precursor ion, MS/MS spectra were collected at fourcollision energies: 18, 27, 35 and 42 V. Dynamic exclusion was enabledfor all experiments for a duration of 78.9 s with a rejection masswindow of m/z 2.3. The ProteinLynx 4.1 software suite (WatersCorporation) was used to de-isotope the MS and MS/MS spectra, and theMS/MS spectra at all four collision energies for each peptide wereaveraged and combined. Processed MS/MS spectra were then searchedlocally against the Swiss-Prot all-species database (downloaded Feb. 24,2010,ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/)using the Mascot 2.2 algorithm (Matrix Science). The local version ofthe Swiss-Prot all-species database was modified to include the sequenceof the recombinant tryptic eEF-2K protein digest analyzed in this work.Peptides with up to three possible missed cleavages were specified as asearch parameter. Protein molecular weight and pI constraints were notused in the database searches. Dynamic chemical modificationscorresponding to M-oxidation, C-carbamidomethylation andS,T,Y-phosphorylation were included as search parameters. A peptide massaccuracy of 150 ppm was used for MS spectra and a peptide fragment massaccuracy of 0.3 Da was used for MS/MS spectra. Peptide summary reportswere generated for all protein identifications using Mascot. Therefore,protein identifications resulted solely from peptide sequenceinformation (MS/MS) without consideration of peptide mass fingerprints(MS). Peptide ion scores equal to or greater than 45-49 (varies for eachdatabase search) represent an identification with 95% confidence (<5%chance that the peptide ID is a random event).

Analysis of the autophosphorylation site mutants—Buffer D was used tomeasure the kinetic activity of the autophosphorylation-site mutants ina reaction containing 2 nM eEF-2K enzyme and 0.5 mM [γ-³²P]ATP (100-1000cpm/pmol) in a final volume of 100 μL. Kinase activity in each case wasdetermined by calculating the rate of phosphorylation of the peptide(μM.s⁻¹) in a similar manner to the general kinetic assay describedabove. The assays were performed in triplicate.

The rate of phosphate incorporation was found to be proportional to theconcentrations of eEF-2K over the entire range of concentrationsexamined (Appendix FIG. 1D). As eEF-2K shows no propensity toself-associate over this concentration range, a mechanism correspondingto more than one eEF-2K molecule in the rate-limiting transition statemay be excluded. Thus, following binding of Ca²⁺/CaM and Mg₂ATP, eEF-2Kis presumed to autophosphorylate in an intramolecular manner (within thesame polypeptide) rather than within an eEF-2K dimer, with regards tothe initial rapid incorporation of the firstmole of phosphate. However,the possibility of the subsequent incorporation of phosphate occurringin an intermolecular manner cannot be ruled out.

Autophosphorylation sites on eEF-2K—To determine the possibleautophosphorylation sites on eEF-2K, the recombinant enzyme was allowedto autophosphorylate in the presence of CaM, Ca²⁺ and Mg₂ATP for 3 h.The sample was resolved by SDS-PAGE and then subjected to in-geldigestion with trypsin or chymotrypsin as described under ‘ExperimentalProcedures’.

Tryptic and chymotryptic digests were then analyzed by tandem massspectrometry and MS/MS spectra from the analysis were searched againstthe modified Swiss-Prot all-species database using Mascot. Peptideidentifications with Mascot scores equal to or above 45 (tryptic digest)or 49 (chymotryptic digest) represent an identification with ≧95%confidence and were considered for protein identification andphosphorylation site determination. Combined data from the analysis ofboth digests gave coverage of ˜86% (624/725) of the eEF-2K sequence,with ˜94% (78/83) of the threonine and serine residues covered (AppendixFIG. 2B). Mass spectrometric analysis of the autophosphorylated sample,the results of which have been summarized in Appendix Table 1, revealedfive sites of autophosphorylation in recombinant human eEF-2K—Thr-348,Thr-353, Ser-445, Ser-474 and Ser-500. MS data also indicated otherresidues (Thr-64 and Ser-491) as being phosphorylated, but thesepeptides did not have significant Mascot scores and hence could not beconfidently claimed as autophosphorylation sites.

The autophosphorylation of the Thr-348 site appears to be critical foractivity of the kinase. Mutation of this site to alanine results in aloss of ˜95% of kinase activity. Negative regulation of eEF-2K activityoccurs through an inhibitory phosphorylation (Ser-78, Ser-359, Ser-366and Ser-396). Regulation through the mTOR pathway involves thephosphorylation of Ser-366 by p70 S6 kinase, and the phosphorylation ofSer-359 and Ser-78 by at least two additional unknown kinases (Wang, X.,et al. EMBO J 2001: 20, 4370-4379; Knebel, A., et al. Biochem. J.2002:367, 525-532; Browne, G. J., and Proud, C. G. Mol. Cell. Biol.2004: 24, 2986-2997). It has been postulated that the Ser-78phosphorylation acts to hinder the binding of CaM to eEF-2K (Browne, G.J., and Proud, C. G. Mol. Cell. Biol. 2004: 24, 2986-2997). Thecdc2-cyclin B complex has been shown to modulate eEF-2K activity viaSer-359 in a manner that is dependent on the cell cycle as well as aminoacid availability, and is perhaps controlled by mTOR (Smith, E. M., andProud, C. G. (2008) cdc2-cyclin B regulates eEF2 kinase activity in acell cycle- and amino acid-dependent manner, EMBO J27, 1005-1016).Regulation through the MAPK cascade occurs via the phosphorylation ofSer-366 by p90^(RSK1) in an ERK-dependent fashion (Wang, X., et al. EMBOJ 2001: 20, 4370-4379). In addition, the stress-activated proteinkinases p38α and p38δ inhibit eEF2K via phosphorylation on Ser-396(Knebel, A., et al. Biochem. J. 2002:367, 525-532). p38δ is also knownto phosphorylate eEF-2K on Ser-359 (Knebel, A., et al. EMBO J 2001:20,4360-4369); (AMPK, PKA, Energy Stress, Elevated cAMP, S398,S500)—involved in positive regulation of eEF-2K activity through anactivating phosphorylation (Ser-398 and Ser-500). Phosphorylation ofSer-398 by the energy-supply regulator AMPK is known to activate eEF-2K(Browne, G. J., et al. Journal of Biological Chemistry 2004: 279,12220-12231). The cAMP-dependent PKA has also been shown to activateeEF-2K via a phosphorylation on Ser-500, and in the process impartsCa²⁺/CaM-independent activity to the kinase (Redpath, N. T., and Proud,C. G. Biochem J 1993: 293 (Pt 1), 31-34; Diggle, T. A., et al. Biochem.J. 2001: 353, 621-626; Diggle, T. A., et al., Biochem. J. 1998: 336,525-529); (Elevated [Ca2+]I, Ca2+/CaM, T248, T252, 5445, 5474, eEF-2Binding Domain)—involved in autophosphorylation of eEF-2K (Thr-348,Thr-353, Ser-445, Ser-474 and Ser-500). Of the 5 autophosphorylationsites, only Thr-348 appears to be essential for activity. Ser-500 is anautophosphorylation site and is also known to be phosphorylated by PKA,and could be the key residue responsible for autophosphorylation-inducedCa²⁺-independence (Mitsui, K., et al. Journal of Biological Chemistry1993: 268, 13422-13433; Redpath, N. T., and Proud, C. G. EuropeanJournal of Biochemistry 1993: 212, 511-520). phosphorylation at Ser-377by MAPKAP-K2.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-20. (canceled)
 21. A method of treating cancer in a patient in needof such treatment, said method comprising administering atherapeutically effective amount of a compound having the formula:

wherein R^(1A) is independently hydrogen, halogen, —CX^(1A) ₃,—C(O)R^(7A), —C(O)—OR^(7A), —C(O)NR^(7A)R^(8A), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(2A) is independently hydrogen, halogen,—CX^(2A) ₃, —C(O)R^(9A), —C(O)—OR^(9A), —C(O)NR^(9A)R^(10A), substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1A) and R^(2A) are optionally joined toform a substituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl; R³ is independently hydrogen, halogen, —CX³ ₃,—CN, —SO₂Cl, —SO₁R¹⁴, —SO_(k)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,—NHC═(O)NR¹¹R¹², —N(O)_(m), —NR¹¹R¹², —C(O)R¹³, —C(O)—OR¹³, —O—C(O)—R¹³,—C(O)NR¹¹R¹², —NR¹¹C(O)R¹³, —OR¹⁴, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;R^(7A), R^(8A), R^(9A), R^(10A), R¹¹, R¹², R¹³, and R¹⁴ areindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; A¹ isindependently ═N— or ═CR³—; k and m are independently 1 or 2; n isindependently an integer from 0 to 4; z1 is independently an integerfrom 0 to 3; and X^(1A), X^(2A), and X³ are independently —Cl, —Br, —I,or —F.
 22. The method of claim 21 having the formula:


23. The method of claim 21 having the formula:


24. The method of claim 21, wherein R^(1A) is independently hydrogen,unsubstituted alkyl, —C(O)R^(7A) or —C(O)—OR^(7A); and R^(7A) isindependently hydrogen, substituted or unsubstituted C₁-C₂₀ alkyl,substituted or unsubstituted 2 to 20 membered heteroalkyl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 3 to 8membered heterocycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, orsubstituted or unsubstituted 5 to 10 membered heteroaryl.
 25. The methodof claim 21, wherein R^(2A) is independently hydrogen, unsubstitutedalkyl, —C(O)R^(9A) or —C(O)—OR^(9A); and R^(9A) is independentlyhydrogen, substituted or unsubstituted C₁-C₂₀ alkyl, substituted orunsubstituted 2 to 20 membered heteroalkyl, substituted or unsubstitutedC₃-C₈ cycloalkyl, substituted or unsubstituted 3 to 8 memberedheterocycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, orsubstituted or unsubstituted 5 to 10 membered heteroaryl.
 26. The methodof claim 21, wherein R³ is independently hydrogen, halogen, —C(O)R¹³,—O—C(O)—R¹³, —C(O)—OR¹³, —OR¹⁴, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. 27.The method of claim 21, wherein R³ is independently hydrogen or —OR¹⁴;and R¹⁴ is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. 28.The method of claim 27, wherein R¹⁴ is independently hydrogen,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl.
 29. The method of claim 27, wherein R¹⁴ is independentlysubstituted or unsubstituted alkyl.
 30. The method of claim 27, whereinR¹⁴ is independently substituted or unsubstituted C₁-C₂₀ alkyl.
 31. Themethod of claim 27, wherein R¹⁴ is independently substituted orunsubstituted C₆-C₁₆ alkyl.
 32. The method of claim 27, wherein R¹⁴ isindependently unsubstituted C₆-C₁₆ alkyl.
 33. The method of claim 21,wherein A¹ is ═CR³—.
 34. The method of claim 21, wherein A¹ is ═N—. 35.The method of claim 21, wherein A¹ is ═CH—.
 36. The method of claim 21,wherein the compound is:


37. The method of claim 21, wherein said cancer is breast cancer,ovarian cancer, pancreatic cancer, liver cancer, glioblastoma, glioma,lung cancer, prostate cancer, leukemia, or melanoma.
 38. The method ofclaim 21, wherein said cancer is breast cancer.
 39. The method of claim21, wherein said cancer is metastatic cancer.
 40. The method of claim21, wherein said compound is co-administered with a chemotherapeuticagent.